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BLA 125742
Toxicology Review of COVID -19 Vaccine (BNT162, PF -07302048)
(Final Report)
From: Nabil, Al Humadi
Through: Martin Green
To: Kirk Prutzman, Ramachandra Naik, Michael Smith
File: BLA 125742, original submission
Product: COVID-19 Vaccine (BNT162, PF -07302048)
Reviewer: Nabil Al-Humadi
BLA sections reviewed:
4.2.3.2 Repeated dose toxicity studies
4.2.3.5. Reproductive and Developmental Toxicity
Type and date of submission: Original, August 31st, 2018
Sponsor: BioNTech RNA Pharmaceuticals GmbH, An der Goldgrube 12 Mainz
Germany 55131
Proposed indication: Prophylactic immunization against COVID -19 in adults ≥18 years
of age
Division name : OVRR/DVRPA
Contents
Précis: ..................................................................................................................................... 4
Introduction: ............................................................................................................................ 5
Proposed clinical st udy: ........................................................................................................... 7
Study number 1: .................................................................................................................... 10
Study number 2: .................................................................................................................... 64
Study number 3 (Reproductive Toxicology Study): ................................................................. 93
Historical data: .................................................................................................................... 117
References: ......................................................................................................................... 123
Table of text tables:
Table 1: Protocol of stability study I for CTM drug substance batches at different storage
conditions .............................................................................................................................. 11
Table 2: Experimental design ................................................................................................. 12
Table 3: Blood sampling schedule for laboratory examinations ............................................... 13
Table 4: Acute phase proteins ................................................................................................ 14
Table 5: Cytokine analysis ..................................................................................................... 14
Table 6: Weighed organs ....................................................................................................... 14
Table 7: Serum chemistry results ............................................................................................ 16
Table 8: D ifferences in albumin and globulin levels and the albumin/ globulin ratio compared to the
control group ......................................................................................................................... 20
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2 Table 9: H ematological results ............................................................................................... 23
Table 10: Test article -related changes in hematological and coagulation parameters for the
treatment with BNT162a1 ...................................................................................................... 25
Table 11: Test article -related changes in hematological and coagulation parameters ................ 26
Table 12: Test article -related changes in hematological and coagulation parameters for the
treatment with BNT162c1 ...................................................................................................... 27
Table 13: test article -related changes in hematological and coagulation parameters .................. 28
Table 14: Acute phase protein levels, day 4 relatives to start date ............................................ 29
Table 15: Acute phase protein levels, day 10 relatives to start date .......................................... 29
Table 16: Acute phase protein levels, day 17 relatives to start date .......................................... 30
Table 17: Cytokine levels in males at study day 1 ................................................................... 31
Table 18: Cytokine levels in males at study day 8 ................................................................... 32
Table 19: Cytokine levels in males at study day 15 ................................................................. 33
Table 20: Cytokine levels in males at study day 17 relatives to start date (48h pa) .................... 34
Table 21: Cytokine levels in females at study day 1 ................................................................ 35
Table 22: Cytokine levels in females at study day 8 ................................................................ 36
Table 23: Cytokine levels in females at study day 15 .............................................................. 36
Table 24: Cytokine levels in females at study day 17 relatives to start date (48h pa) ................. 37
Table 25: Urinalysis results in males at day 10 relatives to start date ....................................... 37
Table 26: Urinalysis results in males at day 17 relatives to start date ....................................... 38
Table 27: Urinalysis results in females at day 10 relatives to start date ..................................... 38
Table 28: Urinalysis results in females at day 17 relatives to start date ..................................... 39
Table 29: Male’s organ weights results. Absolute weights are expressed as mean (grams). Entries
in table are expressed both as organ weight from animals taken at the end of the terminal phase
and recovery phase of the study (main phase organ weight/recovery phase organ weight). ....... 43
Table 30: Female’s organ weight: Absolute weights are expressed as mean (grams). Entries in table are expressed both as organ weight from animals taken at the end of the terminal phase and
recovery phase of the study (main phase organ weight/recovery phase organ weight). .............. 45
Table 31: Male’s gross pathology results. ............................................................................... 46
Table 32: Female’s gross pathology results. ............................................................................ 46
Table 33: Incidences of test article -related microscopic findings for the animals treated with
BNT162a1 ............................................................................................................................ 48
Table 34: Incidences of test article -related microscopic findings for the animals treated ........... 49
Table 35: Incidences of test article -related microscopic findings for the animals treated with
BNT162c1 and BNT162b2 .................................................................................................... 50
Table 36: Microscopic findings at terminal sacrifice ............................................................... 51
Table 37: Test article related effects ....................................................................................... 59
Table 38: Protocol of stability study I for CTM drug substance batches at different storage
conditions .............................................................................................................................. 65
Table 39: parameters evaluated .............................................................................................. 67
Table 40: Clinical laboratory measurements ........................................................................... 68
Table 41: Antibody (Serology) response to vaccine components ............................................. 68
Table 42: Tissue collection, organ weights and tissues processed for slide preparation – Dosing
phase ..................................................................................................................................... 69
Table 43: Tissue collection, organ weights and tissues processed for slide preparation – Recovery
phase ..................................................................................................................................... 71
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3 Table 44: Serum chemistry results for males and females ........................................................ 71
Table 45: Test article -related clinical chemistry parameter effects (mean control values and ratio
relative to control mean) ........................................................................................................ 72
Table 46: Test article -related clinical chemistry parameter effects (mean control values and ratio
relative to control mean) ........................................................................................................ 72
Table 47: Hematology results for males and females ............................................................... 73
Table 48: Test article -related hematology and coagulation parameter effects at main sacrifice
(mean control values and ratio relative to control mean) .......................................................... 75
Table 49: Test article -related hematology and coagulation parameter effects at recovery phase
(mean control values and ratio relative to control mean) .......................................................... 75
Table 50: Male’s organ weight: Absolute weights are expressed as mean (grams). Entries in table
are expresse d as organ weight from animals taken at the end of the terminal phase. ................. 76
Table 51: Female’s organ weight: Absolute weights are expressed as mean (grams). Entries in
table are expressed as organ weight from animals taken at the end of the terminal phase. ......... 77
Table 52: Microscopic findings at terminal sacrifice .............................................................. 82
Table 53: Edema and erythema findings in males at study days 1, 8, and 15 ............................ 84
Table 54: Edema and erythema findings in females at study days 1, 8, and 15 .......................... 85
Table 55: Edema and erythema findings in males and females at recovery phase ..................... 86
Table 56: Geometric mean titers (GMTs) for each dose group by sampling day and sex ........... 87
Table 57: Test item identification ........................................................................................... 93
Table 58: Control item identification ...................................................................................... 93
Table 59: Experimental design of the F0 generation ................................................................ 95
Table 60: General in -life assessments – untreated males and F0 females .................................. 96
Table 61: Geometric mean titer by time -point and by group of females or offspring (fetuses and
pups) ..................................................................................................................................... 99
Table 62: Summary of cohabitation data and maternal performance in littering and Caesarean subsets ................................................................................................................................ 101
Table 63: Mean gravid uterus weight and maternal body weight change ................................ 102
Table 64: Mean Caesarean section data ................................................................................ 104
Table 65: Summary of Foetal External, Visceral and Skeletal Observations ........................... 108
Table 66: Delivery and litter data ......................................................................................... 110
Table 67: Mean pup body weight (grams) ............................................................................. 114
Table 68: Summary of reflex and physical development ........................................................ 114
Table 69: Summary of maternal macroscopic observations ................................................... 115
Table 70: Historical data; Caesarean data collected on day 21 of gestation - page 1/2 .......... 117
Table 71: Historical data; Caesarean data collected on day 21 of gestation - page 2/2 .......... 118
Table 72: Historical data; Malformations (external, internal and skeletal) ............................ 119
Table 73: Historical data; Foetal examination - Fresh visceral examination of body on day 20
or 21 of gestaion................................................................................................................. 120
Table 74: Historical data; Foetal examination – Skeletal examination of body on day 21 of
gestation - Page 1/2 ............................................................................................................ 121
Table 75: Historical data; Foetal examination – Skeletal examination of body on day 21 of
gestation - Page 2/2 ............................................................................................................ 122
Table 76: Historical data; Foetal examination – Skeletal examination of head on day 21 of
gestation ............................................................................................................................. 123
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4 Table of figures :
Figure 1: BioNTech non-clinical platform experience ............................................................... 6
Figure 2: Summary of vaccine dose regimens in the clinical study ............................................. 8
Figure 3: Part A; Dose cohort scheme for uRNA (BNT162a1) and saRNA (BNT162c1) ............ 8
Figure 4: Part A; Dose cohort scheme for modified RNA groups (BNT162b1 and BNT162b2) .. 9
Figure 5: Gamma -glutamyltransferase plasma activity in male rats mean values per group and
standard deviation. TD = Treatment day. ................................................................................ 17
Figure 6: Gamma -glutamyltransferase plasma activity in female rats mean values per group and
standard deviation. TD = Treatment day. ................................................................................ 17
Figure 7: Test article -related changes in plasma activity of gamma-glutamyltransferase compared
to the control group in % ........................................................................................................ 18
Figure 8: Reticulocyte’s levels ............................................................................................... 24
Figure 9: Local reactions ........................................................................................................ 41
Figure 10: Body weight gain of male rats ............................................................................... 42
Figure 11: Body weight gain of female rats ............................................................................ 42
Figure 12: Body temperature of male rats treated once weekly, mean values per group ............ 57
Figure 13: Body temperature of female rats treated once weekly, mean values per group ......... 57
Figure 14: Antibody titer resulting in 50% pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in 50% pse udovirus neutralization (pVN50) are shown by dots;
group mean values are indicated by horizontal bars (±SEM, standard error of the mean). ......... 58
Figure 15: antibody titer resulting in 90% pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in 90% pseudovirus neutralization (pVN90) are shown by dots; group mean values are indicated by horizontal bars (±SEM, standard error of the mean). ......... 58
Figure 16: Mean pup bod weights (g) -Males ......................................................................... 111
Figure 17: Mean pup body weights (g) -Females ................................................................... 111
Précis:
Study number 1:
In this repeat (groups 1 to 5 and 7 animals were dosed by IM on study days 1, 8, and 15 and
group 6 animals were dosed on study days 1 and 8) dose toxicology study, rats were assigned to
7 different groups and treated with control or test article (see experimental design). Animals, 18 per sex per group, were treated with a final dose concentration of 0, 10, 30, or 100 [µg/animal].
Animals were euthanized on study days 10 and 17. Except for group 6 (30 µg/animal [LNP
saRNA RBD] test item 5), immune responses were reported in all other treated groups.
Study number 2:
In this repeat (study days 1, 8, and 15) dose toxicology study, rats were assigned to 3 different
groups and treated with control or test article (see experimental design). Animals, 15 per sex per
group, were treated with a final dose concentration of 30 [µg/animal]. Animals were euthanized on study days 17 and 22. Immune responses were reported in all treated groups.
Study number 3 (Developmental toxicology study):
Animals were randomized and assigned to 4 different groups. Each group consisted of 22
females. Animals were administered 4 doses of saline or test article ( 30 [µg/animal]) on study
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5 day 1 (21 days before mating, M -21) and day 8 (14 days before mating, M -14) and on gestation
days 9 and 20. Animals were euthanized according to the following schedule:
F0 Females: Caesarean subset: On GD21.
Littering subset: After weaning of the F1 pups (females that fail to produce a viable litter
by GD26 will be euthanized and necropsied).
Unmated Females: After completion of the mating period.
Pups: On PND4 (unselected pups) or on PND21.
Introduction:
Coronavirus infection 2019 (COVID -19) are increasing every day and spreading globally,
affecting more and more countries.
The World Health Organization (WHO) characterized the COVID -19 outbreak as pandemic on
March 11th, 2020. At the time of writing this report, more than 15 million people around the
world were affected and more than 600 thousand people were died. Currently, no approved vaccines or antiviral drugs to prevent or treat SARS -CoV-2 infections or its associated disease
COVID-2019 (1).
Significant advantage over more conventional vaccine approaches when using an RNA -based
vaccine encoding a viral antigen that is translated to protein by the vaccinated organism to
induce a protective immune response. RNA vaccines do not carry the risks associated with
infection, unlike live attenuated vaccines. This kind of vaccines may be given to people who cannot be administered li ve virus (such as pregnant women and immunocompromised persons).
The manufacturing of the RNA -based vaccines is via a cell -free in vitro transcription process.
This method allows an easy and rapid production, and the prospect of producing high numbers of vaccination doses within a shorter time period than achieved with conventional vaccine
approaches. In outbreak scenarios, this capability is pivotal to enable the most effective response.
The core innovation of the RNA vaccine is based on in vivo delivery of a pharmacologically
optimized, antigen -encoding RNA to induce robust neutralizing antibodies and a concomitant T
cell response to achieve protective immunization with minimal vaccine doses (2-4).
There are three different RNA platforms under development at BioNTech. These platforms are
nonmodified uridine containing mRNA (uRNA, BNT162a), nucleoside modified mRNA (modRNA, BNT162b), and self -amplifying mRNA (saRNA, BNT162c). In more than a dozen
non-clinical GLP safety studies, all three RNA platforms hav e been tested. As for uRNA and
modRNA, there is pre -existing clinical safety data. These data have been obtained primarily with
RNAs formulated with which are related, but not identical, to those to be used in this trial.
Generated by BioNTech, the non-clinical toxicity data suggest a favorable safety profile for
uRNA and modRNA, as well as saRNA formulated with different nanoparticles for various administration routes, including injection. After dosing, the favorable safety
profile is notable because it results in a higher systemic exposure than the planned IM dosing in
this trial. The findings from this study were mild and mostly related to the mode -of-action and
(b) (4)
(b) (4)
(b) (4)
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6 the RNA -intrinsic stimulation of innate immune sensors. In rodents, the non-clinical safety
profile of uRNA and modRNA was predictive for clinical safety.
Figure 1: BioNTech non-clinical platform experience
Pre-IND meeting was held for this IND on April 06, 2020.
Nonclinical:
Sponsor Question 2:
Does CBER agree that the proposed contents of the nonclinical package, including interim results of the ongoing pivotal GLP rat toxicity study (38166), will be sufficient to support initiation of the planned Phase 1/2 study in the US?
Regarding the ongoing pivotal GLP rat toxicity study (38166), the initial IND will include an interim report with the in -life endpoints (including clinical pathology and partial cytokine
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
(b) (4)
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7 results) from the dosing phase. The dosing phase histology, remaining cytokine results, all
serology results, and all the recovery phase endpoint results will be submitted as soon as they
become available, but no later than 120 days after submission of the IND. Does CBER agree?
FDA Response to Question 2: We agree that th e proposed contents of the nonclinical package, including interim results of the
ongoing pivotal GLP rat toxicity study (38166), will be sufficient to support initiation of the
planned Phase 1/2 study in the US. We also agree to accept an interim report of the in -life
endpoints in the initial IND with the remainder being submitted at a later point in time but no
later than 120 days after submission of the IND.
Proposed clinical study:
The clinical study is a multi-site, phase I/II, 2 -part, dose -escalation trial investigating the safety
and immunogenicity of four prophylactic SARS -CoV-2 RNA vaccines against COVID -2019
using different dosing regimens in healthy adults.
In this study four different vaccines (BNT162a1, BNT162b1, BNT162b2, and BNT162c2) will
be tested. Two parts will be included in this study:
Part A
A dose -finding part with four dose cohorts (treatment groups) for each vaccine and one pre -
defined and one optional dose level for a de -escalation approach. A dose-escalation design will
be followed in the first part of the trial (part A). Subjects in this trial (first-in -human [FIH]
immunization) will be immunized using a sentinel dosing/subject staggering (EMA 2017 guidance “Strategies to Identify and Mitigate Risks for First-in -Human and Early Clinical Trials
with Investigational Medicinal Products”). Th e table below shows the FIH starting dose and the
planned escalation/de-escalation doses:
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8
Figure 2: Summary of vaccine dose regimens in the clinical study
Figure 3: Part A; Dose cohort scheme for uRNA (BNT162a1) and saRNA (BNT162c1 )
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9
Figure 4: Part A; Dose cohort scheme for modified RNA groups (BNT162b1 and BNT162b2)
DL = Dose level; SRC = Sa fety Review Committee.
Figure of graphical depiction of the dose-finding process in part A
Part B Dedicated to recruit expansion cohorts with dose levels which are selected from data generated in part A. Using a P/B regimen, the vaccines BNT162a1, BNT162b1, and BNT162b2 will be
administe red. For the vaccine BNT162c2, SD regimen will be used. After evaluation of
aggregate data from part A, details of part B will be defined using a protocol amendment. Based
on analysis of both immunogenicity and safety data gathered in part A, progression t o part B will
be decided. Immunogenicity and safety will be thoroughly assessed to select the vaccine and the
dose(s) to be further evaluated in part B.
Safety data to be evaluated includes the package used by the SRC to assess individual dose levels. Imm unogenicity of all doses will be assessed. In the protocol amendment, a summary of
relevant safety and tolerability data collected in part A will be included. Also, the protocol amendment will include part B specific inclusion/exclusion criteria, objectives/endpoints, a description of the planned statistical analyses, and descriptions of any added trial assessments and procedures.
The design of part B will be a randomized, placebo -controlled in the likely target population
(e.g., high risk populations such as elderly and/or immunocompromised populations). Part B may
employ a surrogate marker as a measure of vaccine efficacy.
Studies reviewed for this BLA:
1- Repeat -dose toxicity study of three LNP -formulated RNA platforms encoding for viral
proteins by repeated intramuscular administration to Wistar Han rats. Study number:
38166 (submitted in amendment 0).
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10 2- 17-day intramuscular toxicity study of BNT162B2 (V9) and BNT162B3C In Wistar Han
rats with a 3 -week recovery. Study number: 20GR142 (submitted in amendment 32).
3- A Combined Fertility and Developmental Study (Including Teratogenicity and Postnatal
Investigations) of BNT162b1, BNT162b2 and BNT162b3 by the Intramuscular Administration in the Wistar Rat. Study number: 20256434 (submitted in amendment
141).
Studies not reviewed in all amendments:
None.
Toxicology Study Review
Study number 1:
Title and study number: Repeat -dose toxicity study of three LNP -formulated RNA platforms
encoding for viral proteins by repeated intramuscular administration to Wistar Han rats. Study
number: 38166.
Performing laboratory:
St
udy initiation date: March 17, 2020
Final report date : July 1, 2020
Test article batch/lot:
Test Article Batch Number Sta bility
Buffer (PBS/300 mM Sucrose) 090320 23 hours Not reported
RBL063.3" (BNT162a - 1) CoVVAC/090320 Not reported
RBP020.3" (BNT162b - 1) CoVVAC/100320 Not reported
RBP020.1" (BNT162b - 2) CoVVAC/160320 Not reported
RBS004.3" (BNT162c - 1) CoVVAC/130320 Not reported
Animal species and s train : Rat/Wistar/Crl:WI(Han)
Breeder/supplier:
Num
ber
of animal per group and sex: 15/sex/group
Age: Approximately 10 -14 weeks at 1st dosing
Body weight range:
Males: 252.8g -343.9g
Females: 188.3g -267.3g
Route and site of administration: Intramuscular (IM)
Volume of injection: 0.5 mL
Frequency of administration and study duration:
For groups 1 to 5 and 7:
On test days 1, 8 and 15; in total 3 administration days at one -week intervals per
animal.
For group 6:
On test days 1 and 8; in total 2 administration days at one -week interval per animal.
(b) (4)
(b) (4)
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11 Dose : See study design
Stability: Analysis of stability, homogeneity and concentration of the test article under test
condit ions was not performed as part of the study. Stability studies were performed by the
sponsor of the IND. At the time of submitting this study, stability studies with the first clinical trial material batch have just been started. Up to now no results are available. Stability data will be included in any upcoming amendment. The table below shows the protocol of stability study I
for CTM drug substance batches:
condi
tions
Means of administration: Intramuscular (IM)
Report status: Interim report
Experimental design:
Animals were randomized and assigned to 7 different groups. Each group consisted of
18/sex/group. Groups 1 to 5 and 7 animals were dosed by IM on study days 1, 8, and 15. Groups
6 animals were dosed by IM on study days 1 and 8. The details of the study design are listed in
the following table:
Group
Dose level
[µg/animal]
(Test item /
Control) No. and sex of
animals
MS + RP + SA Rat numbe r
MS
RP
SA
1 0
(Buffer)
Control
10 + 5 + 3 m
10 + 5 + 3 f
1-10
16-25
11-15
26-30
211-213
214-216
2 30
(LNP uRNA RBD)
Test item 1
10 + 5 + 3 m
10 + 5 + 3 f
31-40
46-55
41-45
56-60
217-219
220-222
(b) (4)
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12
Group
Dose level
[µg/animal]
(Test item /
Control) No. and sex of
animals
MS + RP + SA Rat numbe r
MS
RP
SA
3 10
(LNP uRNA RBD)
Test item 1
10 + 5 + 3 m
10 + 5 + 3 f
61-70
76-85
71-75
86-90
223-225
226-228
4 30
(LNP modRNA
RBD)
Test item 3
10 + 5 + 3 m
10 + 5 + 3 f
91-100
106-115
101-105
116-120
229-231
232-234
5 100
(LNP modRNA
RBD)
Test item 3
10 + 5 + 3 m
10 + 5 + 3 f
121-130
136-145
131-135
146-150
235-237
238-240
6 30
(LNP saRNA RBD)
Test item 5
10 + 5 + 3 m
10 + 5 + 3 f
151-160
166-175
161-165
176-180
241-243
244-246
7 100
(LNP modRNA
Sp2)
Test item 4
10 + 5 + 3 m
10 + 5 + 3 f
181-190
196-205
191-195
206-210
247-249
250-252
Errone ousl y
treated
a nimals#: 100
(LNP uRNA RBD)
Test item 1
0 + 0 + 3 m
-
-
253-255
m: ma le, f: female , MS: Main study , RP: Recovery period , SA: Sa tellite a nimals for cytokine a nalysis (except la st
group) . #: Due to shortly planned dose reduction of group 3, three animals had already
been dosed as originally planned with 100 µg/animal. These three animals were replaced
by 3 spare animals in group 3. The three erroneously treated animals were maintained for
at least 48 hours as a non-GLP group with observations reported informally to the sponsor
(body weight (test day 1 and 24 and 48 hours post injection), body temperature (24 and 48
hours post injection) and local tolerance (24 and 48 hours post injection)).
Table 2: Experimental design
Methods:
Randomization procedure: Yes
Statistical analysis plan: Yes.
The following parameters were evaluated: Clinical observations (twice daily), local tolerance
[Draize scoring] (4, 24, and 48 hours after each injection), body weights (prior to injection on study days 1, 8, and 15, after treatment on study days 2, 9, and 16, and at necropsy on study days
10 or 17), food consumption (weekly), ophthalmology (before first dosing and at the end of the dosing period), body temperature (4 and 24 hours post injection on study days 1, 8, and 15),
cytokines (study days 1, 8, 10, 15, and 17), clinical chemistry, hematology, coagulation, and
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13 acute phase proteins (study days 4, 10, and 17), urinalysis (study days 10 and 17), serology (day
10 [BNT162c1] or at day 17 after first immunization [BNT162a1, BNT162b1, and BNT162b2]). Postmortem evaluations were performed on study days 10 (groups 6 and 7) and 17 (groups 1 to
5).
Parameters Frequency of Testing
Cageside observation1 Twice daily
Clinical observations 2 Twice daily
Body weight Prior to injection on study days 1, 8, and
15, after treatment on study days 2, 9, and
16, and at necropsy on study days 10 or 17
Food consumption Weekly
Body temperature 4 and 24 hours post injection on study days
1, 8, and 15
Ophthalmologic exam Before first dosing and at the end of the
dosing period
Clinical chemistry* Study days 4, 10, and 17
Hematology* Study days 4, 10, and 17
Coagulation* Study days 4, 10, and 17
Local tolerance [Draize scoring] 4, 24, and 48 hours after each injection
Serology Day 10 (BNT162c1) or at day 17 after first
immunization (BNT162a1, BNT162b1, and
BNT162b2)
Cytokines Study days 1, 8, 10, 15, and 17
Urinalysis Study days 10 and 17
Postmortem study evaluations Study days 10 (groups 6 and 7) and 17
(groups 1 to 5)
* Site collection of blood samples were retrobulbar venous plexus.
Day of sampling Animals Parameters
Test day 4: The first 5 ma in study a n ima ls
per sex and group and a ll
recovery a nimals. Hema tology
Clinica l chemistry
Acute phase proteins
At ma in study termination
(on the day of dissection,
i.e. on test days 10 or 17): All ma in study a nimals Hema tology
Coagulation
Clinica l chemistry
Acute phase proteins
Table 3: Blood sampling schedule for laboratory examinations
1 Cageside observations include mortality, morbidity, gener al health and signs of toxicity.
2 Clinical observations include evaluation of skin and fur, eye and mucous membr anes, respiratory, circulatory,
auto nomic and central nervous systems, somatomotor and behavior.
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14 Parameter Matrix Total amount
of sample Aliquots
prepared Storage
tempera ture ELISA Kit
α1-a cid
gl ycoprotei n
Serum
150 µL
2 x 75 µL
-20 ° C
± 10 % Rat Alpha 1 Acid
Glycoprotein / AGP
ELISA Kit
(ab157729 )
α2
ma croglobulin
Serum
150 µL
2 x 75 µL
-20 ° C
± 10 % Rat alpha 2
Macroglobulin ELISA
Kit (ab157 730 )
Table 4: Acute phase proteins
Cytokines Matrix Total
amount of
sample Aliquots
prepared Storage
tempera ture Method
IFN-γ
TNF-α
IL-1-β
IL-6
IL-10
Serum
150 µL
2 x 75 µL
–20 ° C
± 10 % Cytometric bead array
(Proc artaPlex) usi ng
Cytomics FC 500
(Beckman Coulter GmbH,
47704 Krefeld, Germa ny)
Table 5: Cytokine analysis
Postmortem procedures:
Table of weighed organs
Adrenal gla nd (2) Ovary (2)
Bra in Pituita ry gla nd
Epididymis (2) Prostate
Heart Spleen
Kidney (2) Testicle (2)
Liver Thymus
Lungs Thyroid (1) (including parathyroids)
Lymph nodes (cervical (1), mesenteric (1))
Table 6: Weighed organs
Results:
No test article -related mortality was reported.
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15
Clinical chemistry and hematology:
CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT STUDY DAY
(SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
than 1.5 so indicated otherwise ≥ 1.5)) NOT OF NOTE
ELECTROLYTE BALANCE Calcium, chloride, potassium, sodium,
phos phorus
CARBOHYDRATE
METABOLISM Glucose
LIVER FUNCTION :
A) HEPATOCELLULAR
B) HEPATOBILIARY Alanine aminotransferase (ALT or
SGPT)
SD4 F ↓ = 0.6 G7 Aspartate aminotransferase (AST or
SGOT)
Tota l bilirubin
Alkaline phosphatase (ALP)
ACUTE PHASE REACTANTS Fibrinogen (also under coagulation)
KIDNEY FUNCTION
Crea tinine
Blood Urea Nitrogen (BUN)
OTHERS
(ACID/BASE BALANCE ,
CHOLINESTERASES ,
HORMONES , LIPIDS ,
METHEMOGLOBIN , AND
PROTEINS ) Fa sting triglycerides
SD4 M ↓ = 0.6 G2
SD4 M ↓ = 0.3 G3
SD4 M ↓ = 0.3 G5 SD4 M ↓ = 0.3 G6
SD4 M ↓ = 0.3 G7 SD4 F ↓ = 0.3 G3
SD4 F ↓ = 0.6 G4
SD4 F ↓ = 0.3 G5
SD4 F ↓ = 0.4 G6
SD4 F ↓ = 0.3 G7
SD17 F ↑ = 1.8 G3
Total Cholesterol
SD17 M ↓ = 0.6 G2
SD17 M ↓ = 0.6 G4
Creatine kinase (CK)
SD4 M ↑ = 1.7 G4
Gamma -GT
SD4 M ↑ = 4.4 G2
SD4 M ↑ = 3.1 G3
SD4 M ↑ = 2.7 G4
SD4 M ↑ = 3.5 G5 SD4 M ↑ = 3.8 G6
SD4 M ↑ = 3.4 G7
SD17 M ↑ = 2.7 G2
SD17 M ↑ = 1.9 G3
SD17 M ↑ = 2.2 G4
SD17 M ↑ = 2.6 G5
SD17 M ↑ = 3.0 G7 Albumin (A)
Total protein
Carbon dioxide
Globulin
A/G ratio
FDA-CBER-2021-5683-1150628
BLA 125742
16 CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT STUDY DAY
(SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
than 1.5 so indicated otherwise ≥ 1.5)) NOT OF NOTE
SD4 F ↑ = 4.2 G2
SD4 F ↑ = 3.1 G3
SD4 F ↑ = 2.6 G4
SD4 F ↑ = 4.2 G5
SD4 F ↑ = 4.3 G6
SD4 F ↑ = 4.6 G7
Lactate dehydrogenase (LDH)
SD4 F ↑ = 1.7 G6
Table 7: Serum chemistry results
Clinical chemistry results showed a decrease in ALT levels in group 7 females at study day 4. Triglyceride levels were decreased in groups 2, 3, 5, 6, and 7 males at study day 4. Triglyceride
levels were decreased in groups 3, 4, 5, 6, and 7 females at study day 4. Triglyceride leve ls were
increased in group 3 females at study day 17. Cholesterol levels were decreased in groups 2 and
4 males at study day 17. Creatine kinase levels were increased in group 4 males at study day 4. Gamma -GT levels were increased in groups 2, 3, 4, 5, 6, and 7 males at study day 4. Gamma -GT
levels were increased in groups 2, 3, 4, 5, and 7 males at study day 17. Gamma -GT levels were
increased in groups 2, 3, 4, 5, 6, and 7 females at study day 4. LDH levels were increased in group 6 females at study day 4.
FDA-CBER-2021-5683-1150629
BLA 125742
17 Figure 5: Gamma-glutamyltransferase plasma activity in male rats mean values per group and
standard deviation. TD = Treatment day.
Figure 6: Gamma-glutamyltransferase plasma activity in female rats mean values per group and
standard deviation. TD = Treatment day.
FDA-CBER-2021-5683-1150630
BLA 125742
18 Figure 7: Test article -related changes in plasma activity of gamma-glutamyltransferase compared
to the control group in %
In all test article -treated groups, an increase in albumin plasma levels and a decrease in globulin
plasma levels, resulting in an altered albumin/globulin ratio, were reported. These changes are
consistent with an ac ute phase response in albumin and globulin where albumin goes down and
globulin goes up with inflammation, and the albumin/globulin ratio decreases. The following
table lists the statistically significant changes reported in albumin and globulin levels and the
alb./glob. ratio.
Statistically significant differences in albumin and gl obulin levels and
the albumin/ globulin ratio compared to the control group
Parameter Group Test item Dose
[µg/anima l] Sex Test
day Change
[%]
Albumin 2 BNT162 a1 30 m 4 -9.4**
17 -5.5**
f 4 -14.1**
17 -8.8**
3 BNT162 a1 10 m 4 -6.8**
17 -5.9**
f 4 -11.3 **
17 -8.8**
4 BNT162 b1 30 m 4 -4.1**
17 -3.9**
f 4 -8.4*
17 -9.8**
5 BNT162b1 100 m 4 -7.0**
17 -3.8**
FDA-CBER-2021-5683-1150631
BLA 125742
19 Statistically significant differences in albumin and gl obulin levels and
the albumin/ globulin ratio compared to the control group
Parameter Group Test item Dose
[µg/anima l] Sex Test
day Change
[%]
f 4 -10.8**
17 -10.5**
6 BNT162 c1 30 m 4 -7.7**
f 4 -11.7**
7 BNT162 b2 100 m 4 -9.1**
17 -5.9**
f 4 -12.6**
17 -11.0**
Globulin 2 BNT162a1 30 m 4 +9.5**
17 +9.7**
f 17 +13.6 **
4 BNT162 b1 30 m 4 +15.9 **
17 +18.6 **
m 17 +9.5**
f 17 +17.9 **
5 BNT162 b1 100 m 4 +9.1**
17 +26.3 **
f 17 +14.4**
6 BNT162 c1 30 m 4 +6.5*
7 BNT162 b2 100 m 4 +7.3*
17 +23.1**
f 17 +17.7 **
Alb u m in /Globu lin
Ra tio 2 BNT162 a1 30 m 4 -17.1**
17 -13.9**
f 4 -18.0**
17 -19.3 **
3 BNT162 a1 10 m 4 -8.4**
17 -11.7**
4 BNT162b1 30 m 4 -17.1 **
17 -18.9**
f 4 -16.3**
17 -23.6 **
5 BNT162 b1 100 m 4 -14.6**
FDA-CBER-2021-5683-1150632
BLA 125742
20 Statistically significant differences in albumin and gl obulin levels and
the albumin/ globulin ratio compared to the control group
Parameter Group Test item Dose
[µg/anima l] Sex Test
day Change
[%]
17 -23.8**
f 4 -17.0**
17 -21.7**
6 BNT162 c1 30 m 4 -13.2**
f 4 -10.1**
7 BNT162b2 100 m 4 -15.1 **
17 -23.6**
f 4 -15.7**
17 -24.4 **
m = M a le
f = Fema le
*/** Statistically significan t at p = 0.01 / p = 0.05 (ba sed on numer ical da ta, not on percen t di fference).
Table 8: Differences in albumin and globulin levels and the albumin/ globulin ratio compared to the
control group
HEMATOLOGY
MEASUREMENT
RELATED TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT, STUDY
DAY (SD), SEX, DOSE GROUP (G), DIRECTION, FOLD CHANGE if great
or less than 1.5 3, ie, ≥1.6 or ≤ 1.6 Not of NOTE
Red blood cells
Reticulocytes
SD4 M ↓ = 0.2 G2
SD4 M ↓ = 0.4 G3
SD4 M ↓ = 0.6 G4
SD4 M ↓ = 0.4 G5
SD4 M ↓ = 0.3 G6
SD4 M ↓ = 0.3 G7 SD4 F ↓ = 0.4 G2
SD4 F ↓ = 0.5 G3
SD4 F ↓ = 0.6 G5
SD4 F ↓ = 0.4 G6
SD4 F ↓ = 0.5 G7 Hematocrit (Hct)
Hemoglobin Conc. (Hb)
Mea n Corp. Hb. (MCH)
Mea n Corp. Hb. Conc. (MCHC), Mea n Co rp. Volume (MCV)
Tota l Erythrocyte Count (RBC)
White blood cells
Monocyte count:
SD4 F ↑ = 2.3 G2
SD4 F ↑ = 1.9 G6
SD17 F ↑ = 2.0 G2 SD17 F ↑ = 2.3 G3
SD17 F ↑ = 2.3 G4 Ma crophage
Leukocytes
3 With rounding up at the tenth decimal place. Therefore, 1.54 or less becomes 1.5 and is not reported
and 1.55 or greater becomes 1.6 and is reported.
FDA-CBER-2021-5683-1150633
BLA 125742
21 HEMATOLOGY
MEASUREMENT
RELATED TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT, STUDY
DAY (SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
or less than 1.5 3, ie, ≥1.6 or ≤ 1.6 Not of NOTE
SD17 F ↑ = 2.1 G5
SD17 F ↑ = 1.6 G7
Lymphocyte count
SD17 M ↓ = 0.5 G2
SD1 7 M ↓ = 0.6 G5
SD17 M ↓ = 0.5 G7
Neutrophil count
SD4 M ↑ = 1.8 G2 SD4 M ↓ = 0.6 G5
SD17 M ↑ = 3.0 G2
SD17 M ↑ = 2.3 G3
SD17 M ↑ = 2.5 G4
SD17 M ↑ = 2.9 G5
SD17 M ↑ = 3.2 G7
SD4 F ↑ = 3.5 G2
SD4 F ↑ = 1.6 G5
SD4 F ↑ = 2.0 G6
SD4 F ↑ = 2.3 G7
SD17 F ↑ = 6.9 G2
SD17 F ↑ = 4.4 G3
SD17 F ↑ = 5.9 G4
SD17 F ↑ = 7.4 G5 SD17 F ↑ = 7.8 G7
Eosinophils count
SD4 M ↓ = 0.6 G6 SD17 M ↓ = 0.5 G2
SD17 M ↓ = 0.6 G3
SD17 M ↑ = 1.7 G5
SD17 M ↑ = 2.2 G7
SD17 F ↑ = 1.6 G3
SD17 F ↑ = 3.3 G4
SD17 F ↑ = 5.4 G5
SD17 F ↑ = 6.1 G7
Ba sophils
SD4 M ↑ = 1.8 G3 SD4 M ↑ = 1.6 G5 SD4 M ↑ = 2.3 G6
SD4 M ↑ = 2.5 G7
SD17 M ↑ = 2.1 G2
SD17 M ↑ = 2.3 G3
SD17 M ↑ = 2.0 G4
SD17 M ↑ = 2.1 G5
SD17 M ↑ = 2.5 G7
SD4 F ↑ = 2.2 G2
FDA-CBER-2021-5683-1150634
BLA 125742
22 HEMATOLOGY
MEASUREMENT
RELATED TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT, STUDY
DAY (SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
or less than 1.5 3, ie, ≥1.6 or ≤ 1.6 Not of NOTE
SD4 F ↑ = 1.8 G6
SD4 F ↑ = 1.7 G7
SD17 F ↑ = 3.2 G2
SD17 F ↑ = 2.1 G3
SD17 F ↑ = 2.2 G4
SD17 F ↑ = 2.3 G5
SD17 F ↑ = 2.1 G7
White Blood Cells (WBC)
SD17 M ↑ = 1.6 G2
SD17 M ↑ = 1.6 G3
SD17 M ↑ = 1.6 G4
SD17 M ↑ = 1.8 G5
SD17 M ↑ = 2.2 G7
SD17 F ↑ = 2.0 G2 SD17 F ↑ = 1.6 G3 SD17 F ↑ = 1.8 G4
SD17 F ↑ = 2.0 G5
SD17 F ↑ = 2.1 G7
Large Unstained Cells (LUC)
SD4 M ↑ = 5.6 G2
SD4 M ↑ = 2.2 G3
SD4 M ↑ = 2.2 G5 SD4 M ↑ = 3.2 G6
SD4 M ↑ = 2.8 G7
SD17 M ↑ = 7.1 G2
SD17 M ↑ = 3.4 G3
SD17 M ↑ = 1.7 G4
SD17 M ↑ = 3.5 G5
SD17 M ↑ = 3.4 G7
SD4 F ↑ = 6.7 G2
SD4 F ↑ = 2.1 G3
SD4 F ↑ = 3 .5 G5
SD4 F ↑ = 3.8 G6
SD4 F ↑ = 4.2 G7
SD17 F ↑ = 11.2 G2
SD17 F ↑ = 6.2 G3
SD17 F ↑ = 5.6 G4
SD17 F ↑ = 8.1 G5 SD17 F ↑ = 4.2 G7
Clotting potential
Platelet count
SD17 F ↓ = 0.6 G2
Fibrinogen
SD17 M ↑ = 2.9 G2
SD17 M ↑ = 2.6 G3
SD17 M ↑ = 2.6 G4 Activa ted partial -thromboplastin time
clotting time
Prothrombin time
FDA-CBER-2021-5683-1150635
BLA 125742
23 HEMATOLOGY
MEASUREMENT
RELATED TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT, STUDY
DAY (SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
or less than 1.5 3, ie, ≥1.6 or ≤ 1.6 Not of NOTE
SD17 M ↑ = 2.9 G5
SD17 M ↑ = 3.1 G7
SD17 F ↑ = 2.7 G2
SD17 F ↑ = 2.4 G3
SD17 F ↑ = 2.5 G4 SD17 F ↑ = 2.6 G5
SD17 F ↑ = 2.6 G7
PCT %
SD17 M ↓ = 0.6 G3
SD17 M ↓ = 0.6 G5
SD17 M ↓ = 0.6 G7
SD17 F ↓ = 0.6 G2 SD17 F ↓ = 0.5 G3
SD17 F ↓ = 0.6 G5
SD17 F ↓ = 0.6 G7
Others
Bone marrow cytology
Table 9: Hematological results
Hem
atology results showed decrease in reticulocyte levels in groups 2, 3, 4, 5, 6, and 7 males at
study day 4. Reticulocyte levels were decreased in groups 2, 3, 5, 6, and 7 females at study day 4. Reticulocytes levels were decreased after the 1
st dose but recovered by the end of in-life of the
toxicity study.
FDA-CBER-2021-5683-1150636
BLA 125742
24 Figure 8: Reticulocyte’s levels
Monocyte levels were increased in groups 2 and 6 females at study day 4. Monocyte levels were
increased in groups 2, 3, 4, 5, and 7 females at study day 17. Lymphocyte levels were decreased in groups 2, 5, and 7 males at study day 17. Neutrophil levels wer e increased in group 2 males at
study day 4. Neutrophil levels were decreased in group 5 males at study day 4. Neutrophil levels
were increased in groups 2, 3, 4, 5, and 7 males and females at study day 17. Neutrophil levels
were increased in groups 2, 5, 6, and 7 females at study day 4. Eosinophil levels were decreased in group 6 males at study day 4. Eosinophil levels were decreased in groups 2 and 3 males at study day 17. Eosinophil levels were increased in groups 5 and 7 males at study day 17.
Eosinophi l levels were increased in groups 3, 4, 5, and 7 females at study day 17. Basophil levels
were increased in groups 3, 5, 6, and 7 males at study day 4. Basophil levels were increased in
groups 2, 3, 4, 5, and 7 males at study day 17. Basophil levels were increased in groups 2, 6, and 7 females at study day 4. Basophil levels were increased in groups 2, 3, 4, 5, and 7 females at
study day 17. WBC levels were increased in groups 2, 3, 4, 5, and 7 males and females at study
day 17. LUC levels were increased in groups 2, 3, 5, 6, and 7 males and females at study day 4.
LUC levels were increased in groups 2, 3, 4, 5, and 7 males and females at study day 17.
Platelet count were decreased in group 2 females at study day 17. Fibrinogen levels were
increased in gro ups 2, 3, 4, 5, and 7 males and females at study day 17. PCT% levels were
decreased in groups 3, 5, and 7 males at study day 17. PCT% levels were decreased in groups 2, 3, 5, and 7 females at study day 17.
Groups 2 and 3:
Decreases in the absolute and r elative reticulocyte count, the number of platelets, and red cell
mass, and increases in the numbers of leucocytes, neutrophils, monocytes, large unstained cells
(LUC), basophils and/or the levels of fibrinogen were reported in the test article -treated gro ups.
At the end of the recovery phase, all changes were fully reversed.
FDA-CBER-2021-5683-1150637
BLA 125742
25
Test item-related changes in hematological and coagulation parameters, groups 2
and 3 compared to the control group in %
Parameter BNT162a1
Group 3: 10 µg/animal Group 2: 30 µg/animal
Males Females Males Females
Test day 4
Pla telet s (PLT) None None None -22.7**
Reticulocytes (rela tive) -64.1** -52.0** -75.3** -62.1**
Reticulocytes (absol ut e) -62.1** -51.5** -75.6** -64.4**
Neutrophils (Neut), abs. None None +128.8 ** +245.1 **
Monocyt es (Mono), abs. None None +39.0 +129.5**
Large unclassified cells
(LUC), abs. None None +644.9** +574.7**
Basophils (Baso), abs. None None None +119.2 **
Test day 17
Haemo globin (HGB) None -7.7** None None
Erythrocytes (RBC) None -5.4* None None
Haematocr it (HCT) None -9.7** None None
Leucoc ytes (WBC) None None +79.1 ** +104.1 **
Pla telet s (PLT) -26.1 ** -34.6 ** -26.1 ** -41.7 **
Neutrophils (Neut), abs. +267.1 ** +338.0 ** +430.6 ** +589.4 **
Monoc ytes (Mono), abs. +103.6 ** +131.7 ** +84.7 * +97.4 *
La rge unclassified cells,
(LUC) abs. +455.7** +520.8** +1226.1** +1022.1**
Basophils (Baso), abs. +130.0 ** +105.3 * +110.0 ** +215.8 **
Fibrinogen +155.4** +144.5* +191.3** +174.4**
abs. = absolute
None = No test item-related change.
*/** = Statistically significan t at p ≤ 0.01 / p ≤ 0.05 (based on numer ical data, not on percen t di fference).
Table 10: Test article -related changes in hematological and coagulation parameters for the
treatment with BNT162a1
Groups 4 and 5:
Decreases in the absolute and relative reticulocyte count, the number of platelets, and red cell
mass, and increases in the numbers of leucocytes, neutrophils, monocytes, large unstained cells
(LUC), basophils and/or the levels of fibrinogen were reported in the test article -treated groups.
At the end of the recovery phase, all changes were fully reversed.
FDA-CBER-2021-5683-1150638
BLA 125742
26
Test item-related changes in hematological and coagulation parameters, groups 4
and 5 compared to the control group in %
Parameter BNT162b1
Group 4: 30 µg/animal Group 5: 100 µg/animal
Males Females Males Females
Test day 4
Reticulocytes (r ela tiv e) -43.0 None -65.6 ** -42.6 **
Reticul ocyt es (absol ut e) -44.3 ** None -63.3 ** -42.6 **
Large unclassified cells
(LUC), abs. None None None +250.6**
Test day 17
Haemoglobin (HGB) None -10.5 ** -10.9 ** -13.5 **
Erythrocytes (RBC) None -8.2** -5.6 -9.5**
Haematocr it (HCT) None -9.1** -13.9 ** -14.7 **
Leucocyt es (WBC) None +79.3** +82.2** +102.7**
Platelets (PLT) None None -25.0** -34.4**
Neutrophils (Neut), abs. +304.2** +486.1** +447.3** +636.3**
Monocyt es (M ono), abs. +102.3** +134.4** +77.9* +113.8**
Eosinophils (Eos), abs. +111.9** +227.7** +230.3** +440.4**
La rge unclassified cells
(LUC), abs. +169.3** +457.1** +575.0** +714.3**
Basophils (Baso), abs. +100.0** +121.1** +110.0** +126.3**
Fibrinogen +155.7** +146.2** +192.1** +161.4**
abs. = absolute
None = No test item-related change.
*/** = Statistically significan t at p ≤ 0.01 / p ≤ 0.05 (based on numer ical data, not on percen t di fference).
Table 11: Test article -related changes in hematological and coagulation parameters
for the treatment with BNT162b1
BNT162c1 - G roup 6
Treatment period
Decreases in the absolute and relative reticulocyte count, the number of platelets, and red cell
mass, and increases in the numbers of leucocytes, neutrophils, monocytes, large unstained cells
(LUC), basophils and/or the levels of fibrinogen were reported in the test article -treated groups.
At the end of the recovery phase, all changes were fully reversed.
FDA-CBER-2021-5683-1150639
BLA 125742
27 Test item-related changes in hematological and coagulation parameters, group 6
compared to the control group in %
Parameter Group 6: 30 µg BNT162c1/animal
Males Females
Test day 4
Reticulocytes (r ela tiv e) -76.5** -59.0**
Reticul ocyt es (absol ut e) -74.9** -59.3**
Neutrophils (Neut), abs. +68.4** +104.9**
Monocyt es (M ono), abs. +38.7 +93.7**
Large unclassified cells
(LUC), abs. +360.7** +283.9**
Basophils (Baso), abs. +130.8** None
Test day 10
Haemoglobin (HGB) None ↓
Erythrocytes (RBC) None ↓
Haematocrit ( HCT) None ↓
Leucocytes ( WBC) ↑ ↑
Platelets (PLT) ↓ ↓
Neutrophils (Neut), abs. ↑ ↑
Monocytes (Mono), abs. ↑ ↑
Large unclassified cells
(LUC), abs. ↑ ↑
abs. = absolute. ↑ Increase rela tive to study c ontrol ra nge, but % difference not quanti fia ble due to la cking
conc urren t controls. ↓ Decrease rela tive to study c ontrol ra nge, but % difference not quantif ia ble due to
la cking conc urren t controls. None = No test item-related cha nge. */** = Statistica lly signific ant at p ≤ 0.01 / p
≤ 0.05 (based on numeri cal data, not on percent difference).
Table 12: Test article -related changes in hematological and coagulation parameters for the
treatment with BNT162c1
BNT162b2 - Group 7
Test article -related changes included decreases in the absolute and relative reticulocyte count, the
number of platelets, and red cell mass, and increases in the numbers of leucocytes, neutrophils, monocytes, large unstained cells (LUC), basophils and/or the levels of fibrinogen. All changes fully reversed by the end of the recovery phase.
FDA-CBER-2021-5683-1150640
BLA 125742
28 Test item-related changes in hematological and coagulation parameters, group 7
compared to the control group in %
Parameter Group 7: 100 µg BNT162b2/animal
Males Females
Test day 4
Reticulocytes (rela tive) -74.3** -47.7**
Reticul ocyt es (absol ut e) -72.1 ** -48.2 **
Large uncl a ssified cells
(LUC), abs. +295.5** +319.5**
Basophils (Baso), abs. +150.0** None
Test day 17
Hemo globin (HGB) -9.1** -12.7 **
Erythrocytes (RBC) None -9.8**
Hematocr it (HCT) -11.9** -13.5**
Leucocyt es (WBC) +118.7** +111.0**
Platele ts (PLT) -29.2 ** -34.1 **
Neutrophils (Neut), abs. +605.8 ** +679.8 **
Eosinophils (Eos), abs. +419.3** +509.6**
La rge unclassified cells,
(LUC) abs. +685.2** +594.8**
Basophils (Baso), abs. +146.7 ** +105.3 *
Fibrinogen +205.2 ** +160.2 **
abs. = absolute . None = No test item-related change. */** = Statistically significan t at p ≤ 0.01 / p ≤ 0.05 (based
on numer ical data, not on percen t di fference).
Table 13: test article -related changes in hematological and coagulation parameters
for the treatment with BNT162b2
Acute phase protein levels:
ELISA Parameters -Male ELISA Parameters -Female
Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1] Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1]
Group 1: Mean 64658.6 39774.6 79798.8 18098.2
Control SD 6727.8 3460.7 17269.9 5486.8
N 5 5 5 5
- -
Group 2: Mean 465027.0 ** 727036.0 ** 401386.0** 126189.4**
30 µg/ SD 68141.1 243939.8 32156.3 63343.9
FDA-CBER-2021-5683-1150641
BLA 125742
29 ELISA Parameters -Male ELISA Parameters -Female
Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1] Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1]
a nimal N 5 5 5 5
T. item 1 %Diff 619.2 1727.9 403.0 597.2
Group 3: Mean 304707.0 ** 222958.2 323645.0** 57146.0**
10 µg/ SD 34632.5 118385.8 46893.3 15460.1
a nimal N 5 5 5 5
T. item 1 %Diff 371.3 460.6 305.6 215.8
Group 4: Mean 381868.0 ** 1434571.0 ** 378897.0** 330428.0**
30 µg/ SD 30666.8 522399.7 29869.1 292586.3
a nimal N 5 5 5 5
T. item 3 %Diff 490.6 3506.8 374.8 1725.8
Group 5: Mean 454853.0 ** 2143050.0 ** 444957.0** 1639367.0**
100 µg/ SD 23446.8 71797.8 21643.8 557054.1
a nimal N 5 5 5 5
T. item 3 %Diff 603.5 5288.0 457.6 8958.2
Group 6: Mean 431128.0 ** 685548.0 ** 390580.0** 169592.0**
30 µg/ SD 60320.6 364534.3 23209.4 138784.7
a nimal N 5 5 5 5
T. item 5 %Diff 566.8 1623.6 389.5 837.1
Group 7: Mean 446781.0 ** 2159010.0 ** 445614.0** 1362630.0**
100 µg/ SD 64502.0 78652.0 27975.1 257962.6
a nimal N 5 5 5 5
T. item 4 %Diff 591.0 5328.1 458.4 7429.1
[a] - Anova & Dunnett (Log): ** = p ≤ 0.01
[a1] - Anova & Dunnett (Rank): ** = p ≤ 0.01
Table 14: Acute phase protein levels, day 4 relatives to start date
At study day 4, alpha1 -acid glycoprotein and alpha2 macroglobulin levels were increased
significantly (p ≤ 0.01) in all treated male’s and female’s groups.
ELISA Parameters -Male ELISA Parameters -Female
Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a] Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a]
Group 6: Mean 416278.0 n - 409704.5n -
30 µg/ SD 34413.2 - 31388.8 -
a nimal N 10 - - -
T. item 5 - -
[a] - Anova & Dunnett (Log): ** = p ≤ 0.01
Table 15: Acute phase protein levels, day 10 relatives to start date
FDA-CBER-2021-5683-1150642
BLA 125742
30 ELISA Parameters -Male ELISA Parameters -Female
Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1] Alpha1 -acid
G l ycoprotei n
(ng/mL)
[a] Alpha2
Macrogl ob.
(ng/mL)
[a1]
Group 1: Mean 50334.7 - 52001.7 -
Control SD 11962.9 - 10058.1 -
N 10 - 10 -
- - -
Group 2: Mean 429643.0 ** - 467670.5** -
30 µg/ SD 17527.1 - 35882.2 -
a nimal N 10 - 10 -
T. item 1 %Diff 753.6 - 799.3 -
Group 3: Mean 737003.5 ** - 649429.5** -
10 µg/ SD 124583.7 - 236844.1 -
a nimal N 10 - 10 -
T. item 1 %Diff 1364.2 - 1148.9 -
Group 4: Mean 437627.0 ** - 463014.0** -
30 µg/ SD 54732.7 - 31240.3 -
a nimal N 10 - 10 -
T. item 3 %Diff 769.4 - 790.4 -
Group 5: Mean 970915.5 ** - 980874.0** -
100 µg/ SD 72264.9 - 86180.9 -
a nimal N 10 - 10 -
T. item 3 %Diff 1828.9 - 1786.2 -
Group 7: Mean 1043631.5 ** - 826053.0** -
100 µg/ SD 80157.0 - 274115.3 -
a nimal N 10 - 10 -
T. item 4 %Diff 1973.4 - 1488.5 -
[a] - Anova & Dunnett (Log): ** = p ≤ 0.01
[a1] - Anova & Dunnett (Rank): ** = p ≤ 0.01
Table 16: Acute phase protein levels, day 17 relatives to start date
At study days 10 and 17, alpha1 -acid glycoprotein levels were increased significantly (p ≤ 0.01) in
all treated male’s and female’s groups.
FDA-CBER-2021-5683-1150643
BLA 125742
31 Cytokine levels:
Sex: Male Day 1 Rela tive to Start Da te (PreDs)
Cytokine Levels
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a] IL-6
(pg/m L)
[a] IL-10
(pg/m L)
[a]
Group 1: Mean 7.23 7.10 12.60 3.00 9.90
Control SD 5.60 0.00 0.00 0.00 0.00
N 3 3 3 3 3
- - - - -
Group 2: Mean 4.00 15.50 29.20 3.00n 9.90n
30 µg/ SD 0.00 8.83 26.62 0.00 0.00
a nimal N 3 3 3 3 3
T. item 1 %Diff -44.7 118.3 131.7 0.0 0.0
Group 4: Mean 4.00 7.10 12.60 3.00n 9.90n
30 µg/ SD 0.00 0.00 0.00 0.00 0.00
a nimal N 3 3 3 3 3
T. item 3 %Diff -44.7 0.0 0.0 0.0 0.0
Day: 1 Relative to Start Date (6 h pa)
Group 1: Mean 99.17 66.10 349.93 12.33 212.37
Control SD 7.60 14.69 115.46 8.31 116.87
N 3 3 3 3 3
- - - - -
Group 2: Mean 123.47 87.53 464.57 6.80 157.93
30 µg/ SD 33.70 19.00 114.06 4.59 127.75
a nimal N 3 3 3 3 3
T. item 1 %Diff 24.5 32.4 32.8 -44.9 -25.6
Group 4: Mean 82.40 64.43 347.47 9.20 190.77
30 µg/ SD 11.49 7.01 38.18 1.44 38.89
a nimal N 3 3 3 3 3
T. item 3 %Diff -16.9 -2.5 -0.7 -25.4 -10.2
[a] - Anova & Dunnett
[a1] - Anova & Dunnett(Log)
[a2] - Anova & Dunnett(Rank): n - Ina ppropriate for statistics
Table 17: Cytokine levels in males at study day 1
The levels of IFN -gamma, TNF -alpha, IL -1beta, IL -6, and IL -10 were increased in groups 1, 2,
and 4 males at 6 hours post day 1 treatment.
FDA-CBER-2021-5683-1150644
BLA 125742
32
Sex: Male Day 8 Rela tive to Start Da te (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a] IL-6
(pg/m L)
[a] IL-10
(pg/m L)
[a]
Group 1: Mean 109.77 92.47 447.53 14.57 365.60
Control SD 20.35 19.99 87.14 16.21 74.22
N 3 3 3 3 3
- - - - -
Group 2: Mean 59.07 84.57 432.77 6.67 258.53
30 µg/ SD 50.08 26.63 188.55 3.25 225.11
a nimal N 3 3 3 3 3
T. item 1 %Diff -46.2 -8.5 -3.3 -54.2 -29.3
Group 4: Mean 22.10 22.13 * 93.40 * 3.00 68.67
30 µg/ SD 30.92 26.04 139.95 0.00 101.79
a nimal N 3 3 3 3 3
T. item 3 %Diff -79.9 -76.1 -79.1 -79.4 -81.2
Sex: Male Day 8 Rela tive to Start Date (6 h pa)
Group 1: Mean 88.43 56.80 269.07 4.50 220.07
Control SD 19.95 20.82 111.47 2.60 106.23
N 3 3 3 3 3
- - - - -
Group 2: Mean 117.03 75.83 377.60 3.00 191.67
30 µg/ SD 20.22 18.32 79.12 0.00 56.91
a nimal N 3 3 3 3 3
T. item 1 %Diff 32.3 33.5 40.3 -33.3 -12.9
Group 4: Mean 56.60 41.20 208.17 3.00 84.37
30 µg/ SD 7.54 13.40 74.37 0.00 86.87
a nimal N 3 3 3 3 3
T. item 3 %Diff -36.0 -27.5 -22.6 -33.3 -61.7
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 18: Cytokine levels in males at study day 8
The levels of IFN -gamma, TNF -alpha, IL -1beta, and IL -10 were increased in groups 1, 2, and 4
males at pre-dose and 6 hours post day 8 treatment when compared to day 1. However, the levels of TNF -alpha and IL -1beta decreased significantly in group 4 when compared to group 1 at pre
dose at study day 8. Also, the levels of IFN -gamma and IL -10 decreas ed in group 4 when
compared to group 1 at 6 hours post dose at study day 8.
FDA-CBER-2021-5683-1150645
BLA 125742
33
Sex: Male Day 15 Relative to Start Date (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a]
Group 1: Mean 84.90 66.80 269.17 3.00 178.57
Control SD 61.87 52.44 231.66 0.00 147.46
N 3 3 3 3 3
- - - - -
Group 2: Mean 55.63 87.87 362.97 4.63 167.80
30 µg/ SD 78.85 80.06 383.08 2.83 273.49
a nimal N 3 3 3 3 3
T. item 1 %Diff -34.5 31.5 34.8 54.4 -6.0
Group 4: Mean 44.80 35.77 145.90 3.00 81.00
30 µg/ SD 45.08 46.23 230.88 0.00 123.15
a nimal N 3 3 3 3 3
T. item 3 %Diff -47.2 -46.5 -45.8 0.0 -54.6
Sex: Male Day 15 Relative to Start Date (6 h pa)
Group 1: Mean 125.33 82.30 381.77 3.53 238.63
Control SD 24.16 36.60 149.65 0.92 102.97
N 3 3 3 3 3
- - - - -
Group 2: Mean 190.80 * 112.80 499.80 3.00 270.73
30 µg/ SD 35.23 26.42 83.83 0.00 13.59
a nimal N 3 3 3 3 3
T. item 1 %Diff 52.2 37.1 30.9 -15.1 13.5
Group 4: Mean 124.07 102.80 471.40 5.37 234.17
30 µg/ SD 18.46 27.35 129.00 2.05 107.20
a nimal N 3 3 3 3 3
T. item 3 %Diff -1.0 24.9 23.5 51.9 -1.9
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 19: Cytokine levels in males at study day 15
The levels of IFN -gamma, TNF -alpha, IL -1beta, and IL -10 were increased in groups 1, 2, and 4
males at pre-dose and 6 hours post day 15 treatment when compared to day 1.
FDA-CBER-2021-5683-1150646
BLA 125742
34
Sex: Male Cytokine Levels
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a1]
Group 1: Mean 4.00 7.10 12.60 3.00 9.90
Control SD 0.00 0.00 0.00 0.00 0.00
N 3 3 3 3 3
- - - - -
Group 2: Mean 111.17 ** 25.20 69.83 3.00 9.90
30 µg/ SD 16.10 23.53 84.67 0.00 0.00
a nimal N 3 3 3 3 3
T. item 1 %Diff 2679.2 254.9 454.2 0.0 0.0
Group 4: Mean 31.20 41.97 176.10 7.83 44.70
30 µg/ SD 47.11 60.39 283.19 8.37 60.28
a nimal N 3 3 3 3 3
T. item 3 %Diff 680.0 491.1 1297.6 161.1 351.5
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 20: Cytokine levels in males at study day 17 relatives to start date (48h pa)
The levels of IFN -gamma, TNF -alpha, and IL -1beta were increased in groups 2 and 4 males at
48 hours post day 17 treatment when compared to day 1 and group 1 at study day 17. The levels of IL -10 were increased in group 4 males at 48 hours post day 17 treatment when compared to
day 1 and group 1 at study day 17. IFN -gamma levels were significantly increased in group 2 at
48 hours post day 17 treatment when compared to group 1.
Sex: Female Day 1 Rela tive to Start Da te (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a1] IL-1beta
(pg/mL)
[a1] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a1]
Group 1: Mean 30.67 28.57 119.00 3.00 71.90
Control SD 46.19 23.95 135.10 0.00 107.39
N 3 3 3 3 3
- - - - -
Group 2: Mean 4.00 7.10 12.60 3.00n 9.90
30 µg/ SD 0.00 0.00 0.00 0.00 0.00
a nimal N 3 3 3 3 3
T. item 1 %Diff -87.0 -75.1 -89.4 0.0 -86.2
Group 4: Mean 8.20 7.10 12.60 3.00n 9.90
30 µg/ SD 7.27 0.00 0.00 0.00 0.00
a nimal N 3 3 3 3 3
T. item 3 %Diff -73.3 -75.1 -89.4 0.0 -86.2
Day: 1 Relative to Start Date (6 h pa)
Group 1: Mean 86.50 65.83 345.70 5.77 168.03
Control SD 8.29 29.96 188.07 3.19 78.07
FDA-CBER-2021-5683-1150647
BLA 125742
35 Sex: Female Day 1 Rela tive to Start Da te (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a1] IL-1beta
(pg/mL)
[a1] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a1]
N 3 3 3 3 3
- - - - -
Group 2: Mean 97.87 46.83 246.17 6.07 84.67
30 µg/ SD 32.96 14.73 113.44 4.55 70.78
a nimal N 3 3 3 3 3
T. item 1 %Diff 13.1 -28.9 -28.8 5.2 -49.6
Group 4: Mean 73.37 46.73 235.47 5.50 132.57
30 µg/ SD 29.11 8.39 52.21 4.07 27.24
a nimal N 3 3 3 3 3
T. item 3 %Diff -15.2 -29.0 -31.9 -4.6 -21.1
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 21: Cytokine levels in females at study day 1
Sex: Female Day 8 Rela tive to Start Da te (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a1] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a1]
Group 1: Mean 23.27 12.80 48.37 3.00 17.80
Control SD 31.91 9.87 61.95 0.00 13.68
N 3 3 3 3 3
- - - - -
Group 2: Mean 31.00 27.47 126.83 3.00n 74.30
30 µg/ SD 46.25 35.28 197.86 0.00 111.54
a nimal N 3 3 3 3 3
T. item 1 %Diff 33.2 114.6 162.2 0.0 317.4
Group 4: Mean 54.43 34.17 148.90 3.00n 112.93
30 µg/ SD 39.53 46.88 236.08 0.00 178.46
a nimal N 3 3 3 3 3
T. item 3 %Diff 134.0 166.9 207.9 0.0 534.5
Day 8 Rela tive to Start Date (6 h pa)
Group 1: Mean 77.80 43.67 213.37 3.00 125.70
Control SD 18.19 19.70 99.74 0.00 98.90
N 3 3 3 3 3
- - - - -
Group 2: Mean 103.77 42.77 220.37 3.00n 115.83
30 µg/ SD 53.24 23.93 146.31 0.00 92.56
a nimal N 3 3 3 3 3
T. item 1 %Diff 33.4 -2.1 3.3 0.0 -7.8
Group 4: Mean 80.93 51.47 260.00 3.00n 202.23
30 µg/ SD 30.62 14.82 89.54 0.00 86.64
a nimal N 3 3 3 3 3
FDA-CBER-2021-5683-1150648
BLA 125742
36 T. item 3 %Diff 4.0 17.9 21.9 0.0 60.9
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 22: Cytokine levels in females at study day 8
The levels of IFN -gamma, TNF -alpha, IL -1beta, and IL -10 were increased in groups 2 and 4
females at pre dose at study day 8 treatment when compared to group 1. The levels of IFN -
gamma were increased in group 2 females at 6 hours post dose at study day 8 treatment when
compared to group 1. The levels of IL -10 were increased in group 4 females at 6 hours post dose
at study day 8 treatment when compared to group 1.
Sex: Female Day 15 Relative to Start Da te (PreDs)
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a]
Group 1: Mean 37.33 26.27 116.57 3.00 66.90
Control SD 57.74 33.20 180.08 0.00 98.73
N 3 3 3 3 3
- - - - -
Group 2: Mean 79.63 60.53 252.57 3.00n 148.53
30 µg/ SD 23.68 39.81 182.59 0.00 120.84
a nimal N 3 3 3 3 3
T. item 1 %Diff 113.3 130.5 116.7 0.0 122.0
Group 4: Mean 34.30 7.10 12.60 3.00n 9.90
30 µg/ SD 36.98 0.00 0.00 0.00 0.00
a nimal N 3 3 3 3 3
T. item 3 %Diff -8.1 -73.0 -89.2 0.0 -85.2
Sex: Female Day 15 Relative to Start Date (6 h pa)
Group 1: Mean 121.37 90.97 420.53 3.27 230.10
Control SD 18.61 29.50 143.71 0.46 89.38
N 3 3 3 3 3
- - - - -
Group 2: Mean 185.67 96.20 468.70 3.10 246.37
30 µg/ SD 51.68 23.88 100.85 0.17 46.35
a nimal N 3 3 3 3 3
T. item 1 %Diff 53.0 5.8 11.5 -5.1 7.1
Group 4: Mean 134.57 108.27 504.70 3.67 253.23
30 µg/ SD 23.73 26.68 112.68 0.61 35.48
a nimal N 3 3 3 3 3
T. item 3 %Diff 10.9 19.0 20.0 12.2 10.1
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 23: Cytokine levels in females at study day 15
FDA-CBER-2021-5683-1150649
BLA 125742
37 The levels of IFN -gamma, TNF -alpha, IL -1beta, and IL -10 were increased in group 2 females at
pre dose at study day 15 treatment when compared to group 1. The levels of TNF -alpha, IL -
1beta, and IL -10 were decreased in group 4 females at pre dose at study day 15 treatment when
compared to group 1.
Sex: Female Cytokine Levels
IFN-gamma
(pg/mL)
[a] TNF-al pha
(pg/mL)
[a] IL-1beta
(pg/mL)
[a1] IL-6
(pg/m L)
[a1] IL-10
(pg/m L)
[a]
Group 1: Mean 32.37 20.03 77.83 3.00 45.87
Control SD 49.13 22.40 112.99 0.00 62.30
N 3 3 3 3 3
- - - - -
Group 2: Mean 143.07 * 26.20 97.60 6.10 91.10
30 µg/ SD 28.57 33.08 147.22 5.37 140.64
a nimal N 3 3 3 3 3
T. item 1 %Diff 342.0 30.8 25.4 103.3 98.6
Group 4: Mean 14.73 7.10 12.60 3.00 9.90
30 µg/ SD 18.59 0.00 0.00 0.00 0.00
a nimal N 3 3 3 3 3
T. item 3 %Diff -54.5 -64.6 -83.8 0.0 -78.4
[a] - Anova & Dunnett
[a1] - Anova & Dunnett (Log)
[a2] - Anova & Dunnett (Rank): n - Inappropria te for statistics
Table 24: Cytokine levels in females at study day 17 relatives to start date (48h pa)
The levels of IFN -gamma, IL -1beta, and IL -10 were increased in group 2 females at 48 hours
post study day 17 treatment when compared to group 1. The levels of IFN -gamma, TNF -alpha,
IL-1beta, and IL -10 were decreased in group 4 females at 48 hours post study day 17 treatment
when compared to group 1. Urinalysis:
No test article -related effects on the urinalysis tests were reported.
Sex: Male Urina lysis
Specific
Gra vity
(g/mL)
[a] pH
[a] Urine Volume
- rela tive -
(mL/kg
b.w./24 h)
[a]
Group 6:
30 µg/
ani mal
T. item 5 Mean
SD
N 1.0385 n
0.0130
10
- 6. 94n
0.42
10
- 37.00 n
12.32
10
-
Table 25: Urinalysis results in males at day 10 relatives to start date
FDA-CBER-2021-5683-1150650
BLA 125742
38 Sex: Male Urina lysis
Specific
Gra vity
(g/mL)
[a] pH
[a1] Urine Volume
- rela tive -
(mL/kg
b.w./24 h)
[a1]
Group 1: Mean 1.0309 6.55 45.80
Control SD 0.0057 0.20 5.62
N 10 10 10
- - -
Group 2: Mean 1.0377 6.82 43.72
30 µg/ SD 0.0109 0.40 13.40
a nimal N 10 10 10
T. item 1 %Diff 0.7 4.1 -4.5
Group 3: Mean 1.0355 6.77 38.80
10 µg/ SD 0.0050 0.23 7.32
a nimal N 10 10 10
T. item 1 %Diff 0.4 3.4 -15.3
Group 4: Mean 1.0445 ** 6.62 30.81 **
30 µg/ SD 0.0081 0.26 6.55
a nimal N 10 10 10
T. item 3 %Diff 1.3 1.1 -32.7
Group 5: Mean 1.0458 ** 6.62 33.79 *
100 µg/ SD 0.0145 0.32 9.05
a nimal N 10 10 10
T. item 3 %Diff 1.4 1.1 -26.2
Group 7: Mean 1.0463 ** 6.35 31.67 **
100 µg/ SD 0.0122 0.27 9.65
a nimal N 10 10 10
T. item 4 %Diff 1.5 -3.1 -30.9
Table 26: Urinalysis results in males at day 17 relatives to start date
Specific gravity was increased significantly in groups 4, 5, and 7 males at study day 17. Urine
volume was decreased significantly in groups 4, 5, and 7 males at study day 17.
Sex: Female Urina lysis
Specific
Gra vity
(g/mL)
[a] pH
[a] Urine Volume
- rela tive -
(mL/kg
b.w./24 h)
[a]
Group 6:
30 µg/
ani mal
T. item 5 Mean
SD
N 1.0377 n
0.0130
10
- 6 .4
6n
0.3
3
47.65 n
15.30
10
-
Table 27: Urinalysis results in females at day 10 relatives to start date
FDA-CBER-2021-5683-1150651
BLA 125742
39 Sex: Female Urina lysis
Specific
Gra vity
(g/mL)
[a] pH
[a1] Urine Volume
- rela tive -
(mL/kg
b.w./24 h)
[a1]
Group 1: Mean 1.0349 6.26 45.54
Control SD 0.0047 0.26 10.71
N 10 10 10
- - -
Group 2: Mean 1.0391 6.39 48.55
30 µg/ SD 0.0177 0.28 21.35
a nimal N 10 10 10
T. item 1 %Diff 0.4 2.1 6.6
Group 3: Mean 1.0408 6.27 42.62
10 µg/ SD 0.0129 0.18 13.71
a nimal N 10 10 10
T. item 1 %Diff 0.6 0.2 -6.4
Group 4: Mean 1.0555 ** 6.15 32.31
30 µg/ SD 0.0199 0.28 11.72
a nimal N 10 10 10
T. item 3 %Diff 2.0 -1.8 -29.1
Group 5: Mean 1.0464 6.27 38.55
100 µg/ SD 0.0157 0.21 13.43
a nimal N 10 10 10
T. item 3 %Diff 1.1 0.2 -15.4
Group 7: Mean 1.0400 6.26 38.35
100 µg/ SD 0.0099 0.20 15.62
a nimal N 10 10 10
T. item 4 %Diff 0.5 0.0 -15.8
Table 28: Urinalysis results in females at day 17 relatives to start date
Specific gravity was increased significantly in group 4 fe males at study day 17. Urine volume
was decreased, not to significance, in groups 4, 5, and 7 fe males at study day 17.
Systemic toxicity:
No treatment -related, mortality, nor any toxicologically relevant changes in clinical signs, food
consumption, body temperature, ophthalmic changes, urinalysis, or auditory examination were reported.
Treatment period for BNT162a1 - G
roups 2 and 3
On study days 1, 8, and 15, very slight to moderate edema were reported for all animals following the 1
st, 2nd, and/or 3rd injection of 10 or 30 μg BNT162a1/animal. Following the 1st or
2nd injection (up to 96 h after administration), and/or the 3rd injection (up to 48 h after
administration), male and female animals treated with10 or 30 μg BNT162a1/animal (group 2) revealed very slight to well-defined erythema. At 96 hr’s after the 1
st administration (on study
day 4), all male and female animals of administered 30 μg BNT162a1/animal revealed a scabby skin at the injec tion site. On test day 14 (144 hr’s after the 2
nd administration on test day 8),
severe erythema (grade 4) for 5 of 15 males and 4 of 15 females treated with 10 μg BNT162a1/animal (group 3) was reported. This finding was resolved prior to the 3
rd injection.
Following administration of the 2nd dose on test day 8 (test day 9), the injection site appeared to
FDA-CBER-2021-5683-1150652
BLA 125742
40 be painful for 4 of 15 male animals and 12 of 15 female animals treated with the high dose of 30
μg BNT162a1/animal.
On study day 14 (day before 3rd administration), eschar formation was reported at the injection
site for 5 males and 6 females treated with of 30 μg BNT162a1/animal (group 2). Therefore, on
study day 15, the male animals nos. 32, 34, 37, 39 and 42, and the female animal no. 60 were dosed intramuscularly in the left hind leg instead of the right hind leg as during the previous
administrations.
The macroscopic examination revealed an indurated and/or thickened injection site for all main study animals treated with 30 μg BNT162a1/animal and for the majority of animals treated with
10 μg BNT162a1/animal. For a few animals, an incrustation was reported at the injection site
(high dose: 2 males and 2 females, low dose: one male).
Based on the increase’s incidence and/or severity reported compared with buffer controls, all
findings described above are considered to be test article-related.
Treatment period for BNT162b1 - G
roups 4 and 5
On test days 1, 8, and/or 15, very slight (mostly) to moderate (rarely) edema were reported for all animals following the 1
st, 2nd, and/or 3rd injection of 30 or 100 μg BNT162b1/animal. In the high
dose group (group 5), the two injection sites were occasionally affected to a different degree.
Reported only at 24 hr’s following injection, individual animals of the low dose group treated with 30 μg BNT162b1/animal ( group 4) revealed very slight erythema. These findings were not
dose dependence. On study day 14 (144 hr’s after the 2
nd administration on study day 8), severe erythema (grade 4)
for 3 female animals treated with 100 μg BNT162b1/animal (group 5) was reported. This finding
is considered test article -related. Prior to the 3rd injection, this observation was no longer present.
At macroscopic examination, an indurated and/or thickened injection site was reported for 7
males and 6 females animals per group for the main study animals treated with 30 or 100 μg
BNT162b1/animal.
Treatment period for BNT162c1 - Group 6
On test days 1 and/or 8, very slight (mostly) to moderate (rarely) edema were reported for all animals following the 1
st and/or 2nd injection of 30 μg BNT162c1/animal. Observed only at 96
hr's after the 1st injection, individual male and female animals also revealed very slight erythema.
After the first injection (test day 7), all effects had subsided by 144 hr’s.
The macroscopic examination revealed an indurated and/or thickened injection site for all male
and female main study animals treated with 30 μg BNT162c1/animal. In addition, an incrustation was reported at injection site of one male and one female animal.
Treatment period for BNT162b2 - Gro up 7
On study days 1, 8, and/or 15, very slight to severe (rarely) edema were reported for all animals following the 1
st, 2nd, and/or 3rd injection of 100 μg BNT162b2/animal. All edema reported after
the 1st or 2nd injection had subsided by 96 hr’s post administration. In addition, a few female
animals revealed very slight erythema following 24 to 96 hr’s following the 1st or 2nd injection.
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41 Skin reddening (scored as "severe" erythema) was reported in individual male and female
animals at 144 hr’s after the 2nd injection only but was resolved prior to the 3rd injection.
The macroscopic inspection at necropsy revealed an indurated and/or thickened injection site for
7 of 10 male and 9 of 10 female main study animals treated with 100 μg BNT162b2/animal.
Figur e 9: Local reactions
Local reactions were slight after first immunization but more pronounced after boost with a
reduced immunization interval.
Histopathological examination of injection sites at treatment period
Characterized mostly by moderate inflammation (up to marked) in males and moderate
inflammation in females, the histopathological examination revealed test article -related injection
site findings in all groups. The most severe findings were reported consis tently in animals
administered 100 μg BNT162b1/animal and 100 μg BNT162b2/animal, followed by animals administered 30 μg BNT162a1/animal. The inflammation was characterized by infiltrates of macrophages, granulocytes, and lymphocytes into the muscle, and variably into the dermis and subcutis. Injection site inflammation was associated with mostly moderate edema, mostly mild
myofiber degeneration, occasional muscle necrosis, and mostly mild fibrosis. Skin ulceration
(mild and moderate) was reported in some m ales and females administered either 10 or 30 μg
BNT162a1/animal and one animal administered 30 μg BNT162c1/animal. Inflammation extended into tissues adjacent to the injection site, including mammary tissue, perineural tissue
of sciatic nerve, tissue arou nd the femur / knee and to the draining lymph node (iliac). No
notable injection site findings in the control group was reported.
Body weight gain:
Test article -related treatment decreases in male’s body weight gains were reported in all groups.
In fema les, this effect was less severe in groups 2, 3, 4, and 5. The decrease in groups 6 and 7
body weight gains were higher. The results of the body weight gains are reported in the figures below.
FDA-CBER-2021-5683-1150654
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42 Figure 10: Body weight gain of male rats
Figure 11: Body weight gain of female rats
Figure of body weight gain of male rats treated once weekly,
mean values per group and standard deviation
Group 1: Control
Group 2: 30 µg BNT162a1/animal
Group 3: 10 µg BNT162a1/animal
50
Group 4: 30 µg BNT162b1/animal
Group 5: 100 µg BNT162b1/animal
Group 6: 30 µg BNT162c1/animal
Group 7: 100 µg BNT162b2/animal
40
30
20
10
0
-10
Group:
1
2 3 4 5 6 7
1 2 3 4 5 6 7
Treatment (n = 15), Recovery (n = 5),
TD 1 to TD 16 (TD 9 for group 6) TD 16 to TD 37 (TD 9 to TD 30 for group 6)
Figure of body weight gain of female rats treated once weekly,
mean values per group and standard deviation
Group 1: Control
Group 2: 30 µg BNT162a1/animal
Group 3: 10 µg BNT162a1/animal
50
Group 4: 30 µg BNT162b1/animal
Group 5: 100 µg BNT162b1/animal
Group 6: 30 µg BNT162c1/animal
Group 7: 100 µg BNT162b2/animal
40
30
20
10
0
-10
Group:
1
2 3 4 5 6 7
1 2 3 4 5 6 7
Treatment (n = 15), Recovery (n = 5),
TD 1 to TD 16 (TD 9 for group 6) TD 16 to TD 37 (TD 9 to TD 30 for group 6)
Body weight gain [%]
Body weight gain [%]
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43 Organ Weight:
SEX Males SD (10/17)
GROUPS
1
(CONTROL)
2
3
4
5
6
7
NUMBER OF
ANIMALS 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a]
BODY WEIGHT
(terminal) NC/327 NC/272 NC/328 NC/303 NC/309 272/ NC NC/299
BRAIN NC/2.00 NC/1.95 NC/1.98 NC/1.96 NC/1.98 1.96/NC NC/1.94
ADRENALS -LEFT NC/0.038 NC/0.042 NC/0.041 NC/0.042 NC/0.043 0.040/NC NC/0.043
ADRENALS -RIGHT NC/0.035 NC/0.041 NC/0.041 NC/0.039 NC/0.043 0.039/NC NC/0.037
EPIDIDYMIDES -L NC/0.457 NC/0.449 NC/0.577** NC/0.490 NC/0.53 0.459/NC NC/0.55*
EPIDIDYMIDES -R NC/0.419 NC/0.439 NC/0.524** NC/0.462 NC/0.54** 0.468/NC NC/0.51*
HEART NC/1.14 NC/1.10 NC/1.16 NC/1.10 NC/1.17 1.09/NC NC/1.14
KIDNEYS -L NC/1.426 NC/1.309 NC/1.430 NC/1.404 NC/1.483 1.351/NC NC/1.390
KIDNEYS -R NC/1.479 NC/1.334 NC/1.461 NC/1.418 NC/1.466 1.343/NC NC/1.431
LIVER NC/13.02 NC/10.58* NC/12.78 NC/11.72** NC/13.18 11.03/ NC NC/12.16
LUNGS NC/1.936 NC/1.853 NC/2.019 NC/1.787 NC/1.912 1.702/NC NC/1.877
CERV LYMPH
NODES NC/0.021 NC/0.020 NC/0.019 NC/0.023 NC/0.019 0.017/NC NC/0.016
INGUINAL LYMPH
NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
MANDIBULAR
LYMPH NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
MESENTERIC
LYMPH NODES NC/0.033 NC/0.039 NC/0.040 NC/0.033 NC/0.044 0.033/NC NC/0.050
POPLITEAL LYMPH
NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
PROSTATE NC/0.927 NC/0.789 NC/0.878 NC/0.847 NC/0.790 0.779/NC NC/0.813
SPLEEN NC/0.838 NC/0.976 NC/1.079** NC/0.951 NC/1.030** 1.024/NC NC/1.049**
TESTES -L NC/1.80 NC/1.74 NC/1.87 NC/1.79 NC/1.84 1.75/NC NC/1.84
TESTES -R NC/1.78 NC/1.76 NC/1.86 NC/1.77 NC/1.80 1.78/NC NC/1.82
PITUITARY NC/0.013 NC/0.012 NC/0.013 NC/0.013 NC/0.012 0.010/NC NC/0.011
THYROID and
PARATHYROID NC/0.013 NC/0.014 NC/0.012 NC/0.013 NC/0.011 0.011/NC NC/0.011
THYMUS NC/0.538 NC/0.463 NC/0.527 NC/0.468 NC/0.435 0.465/NC NC/0.388**
OVARIES
UTERUS
NC = Not collected. L = Left; R = Right. CERV = Cervical. [a] - Anova & Dunnett: * = p ≤ 0.05;
** = p ≤ 0.01
Table 29: Male’s organ weights results. Absolute weights are expressed as mean (grams). Entries
in table are expressed both as organ weight from animals taken at the end of the terminal phase
and recovery phase of the study (main phase organ weight/recovery phase organ weight).
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44 Study day 17 male’s results:
Body weight was decreased 17% in group 2. Left adrenal weight was increased 11% in groups 2 and 4. Right adrenal weight was increased 13% in groups 5 and 7. Right adrenal weight was
increased 17% in groups 2 and 3. Right adrenal weight was increased 11% and 23% in groups 4
and 5, respectively. Left epididymides weight was increased 26%, 16%, and 20% in groups 3, 5, and 7, respectively. Right epididymides weight was increased 25%, 10%, 29%, and 22% in groups 3, 4, 5, and 7, respectively. Liver weight was decreased 19% in group 2. Cervical lymph
node’s weight was decreased 24% in group 7. Mesenteric lymph node’s weight was increased
18%, 22%, 33%, and 52% in groups 2, 3, 5, and 7, respectively. Prostate weight was decreased 15%, 15%, and 12% in group 2, 5, and 7, respectively. Spleen weight was increased 16%, 29%, 13%, 23%, and 25% in groups 2, 3, 4, 5, and 7, respectively. Thymus weight was decreased
14%, 13%, 19%, and 28% in groups 2, 4, 5, and 7, respectively.
SEX Females SD (10/17)
GROUPS
1
(CONTROL)
2
3
4
5
6
7
NUMBER OF
ANIMALS 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a]
BODY WEIGHT
(terminal) NC/221 NC/208 NC/217 NC/231 NC/231 195/NC NC/219
BRAIN NC/1.86 NC/1.84 NC/1.85 NC/1.88 NC/1.87 1.78/NC NC/1.87
ADRENALS -L NC/0.045 NC/0.047 NC/0.045 NC/0.046 NC/0.052 0.044/NC NC/0.049
ADRENALS -R NC/0.044 NC/0.046 NC/0.046 NC/0.045 NC/0.051 0.041/NC NC/0.049
EPIDIDYMIDES -L
EPIDIDYMIDES -R
HEART NC/0.914 NC/0.863 NC/0.862 NC/0.952 NC/0.879 0.781/NC NC/0.866
KIDNEYS -L NC/0.938 NC/0.991 NC/0.983 NC/0.998 NC/1.044 0.884/NC NC/1.009
KIDNEYS -R NC/0.989 NC/1.019 NC/0.988 NC/1.027 NC/1.079 0.923/NC NC/1.057
LIVER NC/8.35 NC/9.12 NC/8.82 NC/9.67** NC/10.07** 8.08 /NC NC/9.95**
LUNGS NC/1.333 NC/1.489 NC/1.494 NC/1.565 NC/1.494 1.452/NC NC/1.524
CERV LYMPH
NODES NC/0.016 NC/0.018 NC/0.019 NC/0.018 NC/0.017 0.014/NC NC/0.017
INGUINAL LYMPH
NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
MANDIBULAR
LYMPH NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
MESENTERIC
LYMPH NODES NC/0.034 NC/0.028 NC/0.039 NC/0.033 NC/0.043 0.022/NC NC/0.037
POPLITEAL
LYMPH NODES NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
PROSTATE
SPLEEN NC/0.595 NC/0.941** NC/0.734 NC/0.777* NC/0.925** 0.762/NC NC/0.957**
TESTES -L
TESTES -R
PITUITARY NC/0.015 NC/0.013 NC/0.015 NC/0.014 NC/0.014 0.012/NC NC/0.014
THYROID and
PARATHYROID NC/0.013 NC/0.012 NC/0.011 NC/0.009 NC/0.009 0.009/NC NC/0.011
THYMUS NC/0.456 NC/0.435 NC/0.487 NC/0.457 NC/0.387 0.355/NC NC/0.390
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45 SEX Females SD (10/17)
GROUPS
1
(CONTROL)
2
3
4
5
6
7
NUMBER OF
ANIMALS 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a] 10/10
[a]
OVARY -L NC/0.054 NC/0.056 NC/0.058 NC/0.056 NC/0.055 0.044/NC NC/0.049
OVARY -R NC/0.058 NC/0.054 NC/0.061 NC/0.053 NC/0.059 0.046/NC NC/0.056
UTERUS NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC NC/NC
NC = Not collected. L = Left; R = Right. CERV = Cervical. [a] - Anova & Dunnett: * = p ≤ 0.05;
** = p ≤ 0.01
Table 30: Female’s organ weight: Absolute weights are expressed as mean (grams). Entries in
table are expressed both as organ weight from animals taken at the end of the terminal phase and
recovery phase of the study (main phase organ weight/recovery phase organ weight).
Study day 17 female’s results:
Left and right adrenal weight was increased 16% in group 5. Left kidney weight was increased
11% in group 5. Liver weight was increased 16%, 21%, and 19% in groups 4, 5, and 7,
respectively. Lungs weight was increased 12%, 12%, 17%, 12%, and 14% in groups 2, 3, 4, 5, and 7, respectively. Cervical lymph node’s weight was increased 13%, 19%, and 13% in groups
2, 3, and 4, respectively. Mesenteric lymph node’s weight was decreased 18% in group 2.
Mesenteric lymph node’s weight was increased 15% and 26% in groups 3 and 5, respectively. Spleen weight was increased 58%, 23%, 31%, 55%, and 61% in groups 2, 3, 4, 5, and 7, respectively. Thyroid weight was decreased 15%, 31%, 31%, and 15% in groups 3, 4, 5, and 7,
respectively. Thymus weight was decreased 15% and 14% in groups 5 and 7, respectively.
Gross pathology:
Test article -related findings in all groups included injection site findings, enlarged iliac lymph
nodes, and enlarged spleen. All other findings were considered incidental.
Groups Findings
1M Emphyse matous -lungs (1/10); reddened thymus (1/10)
2M Indurated injections site I (9/10); enlarged spleen (2/10); thickened injections
site I (4/10); enlarged iliac lymph nodes (1/10); reduced in size prostate (1/10); reduced in size seminal vesicles (1/10); enlarged adrenals (1/10); incrusted injections site I and II (2/10); thickened muscle at injections site I (2/10)
3M Indurated injections site I (4/10); enlarged iliac lymph nodes (4/10); enlarged
spleen (5/10); indurated muscle at injections site I (3/10); thickened injections
site I (3/10); skin incrusted (1/10)
4M Indurated injections site I (4/10); enlarged iliac lymph nodes (6/10); enlarged
spleen (1/10); indurated muscle at injections site I (2/10); thickened indurated
muscle at injections site I (1/10)
5M Enlarged injections site I+II (1/10); indurated injections site I+II (2/10);
enlarged iliac lymph nodes (7/10); enlarged spleen (5/10); indurated muscle at
injections site I+II (3/10); thickened injection sites I+II (2/10); en larged
adrenals (2/10)
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46 Groups Findings
6M Indurated injections site I (4/10); enlarged iliac lymph nodes (1/10); enlarged
spleen (5/10); indurated muscle at injections site I (6/10); thickened injection
sites I (9/10)
7M Indurated injections site I+II (5/10); enlarged iliac lymph nodes (5/10);
enlarged renal lymph nodes (1/10); enlarged spleen (2/10); thickened injection
sites I+II (1/10)
Table 31: Male’s gross pathology results.
Groups Findings
1F No findings
2F Incrusted injections site I (1/10); indurated injections site I (3/10); enlarged
iliac lymph nodes (1/10); enlarged spleen (4/10); indurated muscle at injections
site I (4/10); indurated muscle thickened at injections site I (3/10); thickened injection sites I (4/10 ); dilated uterus (1/10); skin incrusted (1/10)
3F Enlarged injections site I (1/10); indurated injections site I (5/10); enlarged
iliac lymph nodes (3/10); enlarged spleen (2/10); indurated muscle at injections site I (1/10); thickened injection sites I (1/10); enlarged adrenals (1/10); dilated uterus [filled with clear liquid] (3/10)
4F Indurated injections site I (5/10); enlarged iliac lymph nodes (4/10); enlarged
spleen (1/10); dilated uterus (1/10); thickened indurated muscle at injection sites I (1/10)
5F Indurated injections site I+II (2/10); indurated muscles at injections sites I+II
(2/10); enlarged iliac lymph nodes (8/10); enlarged spleen (7/10); indurated muscle at injections site I (1/10); thickened injection sites I+II (2/10); sciatic nerve adhered to injection site I (1/10)
6F Indurated injections site I (2/10); enlarged iliac lymph nodes (2/10); enlarged
spleen (1/10); indurated muscle at injections site I (7/10); thickened injection sites I (9/10); incrusted injection site I (1/10)
7F Indurated injections site I (3/10); indurated injections site I+II (4/10); enlarged
iliac lymph nodes (7/10); enlarged spleen (7/10); thickened injection sites I (2/10); muscle jellied [adhered to sciatic nerve and bone] at injection site I (1/10); dil ated uterus [filled with clear liquid] (1/10); sciatic nerve adhered to
injection site I (2/10)
Table 32: Female’s gross pathology results.
Microscopic findings:
Terminal sacrifice
Inflammation at the injection site and surrounding tissues, increased cellularity of germinal
centers and increased plasma cells in the draining (iliac) lymph node, increased cellularity (hematopoiesis) in the bone marrow and spleen, and vacuolation of hepatocytes in the portal regions were the test article -related microscopic findings reported at the end of dosing period. At
the end of the 3-week recovery phase, all microscopic findings were partially or fully recovered.
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47 In all groups, test article -related injection site reactions were reported. Site reactions were mostly
characterized by moderate inflammation (up to marked) in males and moderate inflammation in
females. In groups 5 and 7 (100 μg BNT162b1/animal and 100 μg BNT162b2/animal), the most
severe findings were consistently reported. F ollowed by the animals administered 30 μg
BNT162a1/animals. The inflammation at the injection site was characterized by infiltrates of
macrophages, granulocytes, and lymphocytes into the muscle, and variably into the dermis and subcutis. Injection site inflammation was associated with mostly moderate edema, mostly mild
myofiber degeneration, occasional muscle necrosis, and mostly mild fibrosis. In some males and
females treated with either 10 or 30 μg BNT162a1/animal and one animal administered 30 μg BNT162c1/animal, skin ulceration (mild and moderate) was reported. At the end of the 3-week recovery phase, injection site findings were partially recovered. The inflammation at the
injection sites were extended into tissues adjacent to it. The adjacent tissues included mammary
tissue, perineural tissue of sciatic nerve, tissue around the femur/knee and to the draining lymph node (iliac). At the end of the 3-week recovery phase, these findings were mostly recovered.
In the draining (iliac) lymph node, test artic le-related findings were characterized by increased
cellularity of the follicular germinal centers and increased plasma cells (plasmacytosis) and were
variably present in all groups. In all test article -treated groups, minimal to mild increases in the
cellularity of bone marrow were reported. They were likely secondary to inflammation-related
platelet activation and consumption. Also, extramedullary hematopoiesis in the spleen were reported. A test article -related vacuolation of hepatocytes in the portal regions of the liver was
reported in all groups.
A few other minor microscopic changes were recorded for other organs and were not considered
test article -related. All changes are regarded to be spontaneous in nature being within the normal
background pathology commonly reported in rats of this strain and age.
Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 17
Organ / Finding BNT162 a1
Group 3: 10 µg/animal Group 2: 30 µg/animal
Males Females Males Females
Bone marrow:
- Increased cellulari ty
10/10**
10/10**
10/10**
10/10**
Injection site I (left):
10/10**
10/10**
10/10**
10/10**
- Fibrosis intramuscula r/interstitial
- Fibrosis inter-/perimuscula r 10/10** 10/10** 10/10** 10/10**
- Inflammation, mixed. 10/10** 10/10** 10/10** 10/10**
- Myof iber degener ation 9/10** 9/10** 9/10** 9/10**
- Oedema , subcut is 10/10** 9/10** 6/10* 10/10**
- Oedema intramuscula r/interstitial 7/10** 8/10** 2/10 10/10**
- Oedema inter-/ perimuscula r 10/10** 10/10** 7/10** 10/10**
- Hype rpla sia , epide rmis 9/10** 7/10** 10/10** 9/10**
Surrounding ti ssue of injection sites:
0/10
1/10
3/10
0/10
Perineural tissue of sciatic nerve:
FDA-CBER-2021-5683-1150660
BLA 125742
48 Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 17
Organ / Finding BNT162 a1
Group 3: 10 µg/animal Group 2: 30 µg/animal
Males Females Males Females
- Inflammation (perineural)
Bone, os femori s with joint (surrounding
tissue):
- Inflammation 0/10 1/10 0/10 1/10
Ma m mar y gland (Interstitial tissue):
- Inflammation 0/10 3/10 0/10 0/10
Lymph node (ilia c):
7/10**
7/10**
5/10*
3/10
- Plasmacytosis
- Inflammation 0/10 3/10 5/10* 6/10*
- Increased cellularity, germinal cente r 9/10 10/10** 9/10 8/10
Spleen:
- Increased haematopoiesis
3/10
2/10
0/10
0/10
Liver
- Vacuolation, hepatocellular , peripor tal 1/10 6/10* 1/10 10/10**
…/... number of animals affected per number of animals examined
* signific antly different from control (p ≤ 0.05)
** signific antly different from control (p ≤ 0.01)
Table 33: Incidences of test article -related microscopic findings for the animals treated with
BNT162a1
Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 17
Organ / Finding BNT162 b1
Group 4: 30 µg/animal Group 5: 100 µg/animal
Males Females Males Females
Bone marrow:
- Increased cellulari ty
10/10**
10/10**
10/10**
10/10**
Injection site I and/or II (left/right):
9/10**
10/10**
10/10**
10/10** - Fibrosis intramuscula r/interstitial
- Fibrosis inter-/perimuscula r 9/10** 10/10** 10/10** 10/10**
- Inflammation, mixed. 10/10** 10/10** 10/10** 10/10**
- Myof iber degener ation 9/10** 10/10** 10/10** 10/10**
- Oedema , subcut is 9/10** 10/10** 10/10** 10/10**
- Oedema intramuscula r/interstitial 8/10** 9/10** 10/10** 10/10**
- Oedema inter-/ perimuscula r 10/10** 10/10** 10/10** 10/10**
- Hype rpla sia , epide rmis 9/10** 8/10** 10/10** 10/10**
Surrounding ti ssue of injection sites:
1/10
4/10
7/10**
10/10**
Perineural tissue of sciatic nerve:
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49 Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 17
Organ / Finding BNT162 b1
Group 4: 30 µg/animal Group 5: 100 µg/animal
Males Females Males Females
- Inflammation (perineural)
Bone, os femori s with joint (surrounding
tissue):
- Inflammation 0/10 0/10 4/10 6/10*
Ma m mar y gland (Interstitial tissue):
- Inflammation 0/10 0/10 2/10 1/10
Lymph node (ilia c):
9/10**
8/10**
8/10**
10/10**
- Plasmacytosis
- Inflammation 0/10 0/10 5/10* 8/9**
- Increased cellularity, geminal center 10/10 8/10 10/10 10/10**
Spleen:
- Increased haematopoiesis
0/10
0/10
2/10
7/10**
Liver
- Vacuolation, hepatocellular , peripor tal 0/10 10/10** 8/10** 10/10**
…/... number of animals affected per number of animals examined
* signific antly different from control (p ≤ 0.05)
** signific antly different from control (p ≤ 0.01)
Table 34: Incidences of test article -related microscopic findings for the animals treated
with BNT162b1
Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 10 (group 6) or test day 17 (group 7)
Organ / Finding BNT162 c1 BNT162 b2
Group 6: 30 µg/animal Group 7: 100 µg/animal
Males Females Males Females
Bone marrow:
- Increased cellulari ty
10/10**
10/10**
10/10**
10/10**
Injection site I and/or II (left/right):
9/10**
10/10**
10/10**
10/10**
- Fibrosis intramuscula r/interstitial
- Fibrosis inter-/perimuscula r 9/10** 10/10** 10/10** 10/10**
- Inflammation, mixed 9/10** 10/10** 10/10** 10/10**
- Myof iber degener ation 8/10** 9/10** 10/10** 10/10**
- Oedema , subcut is 9/10** 10/10** 10/10** 10/10**
- Oedema intramuscula r/interstitial 9/10** 10/10** 10/10** 10/10**
- Oedema inter-/ perimuscula r 9/10** 10/10** 10/10** 10/10**
- Hype rpla sia , epide rmis 9/10** 10/10** 9/10** 10/10**
Surrounding tissue of injection sites:
FDA-CBER-2021-5683-1150662
BLA 125742
50 Incidences of test item -related microscopic findings in male and female main study
animals after terminal sacrifice on test day 10 (group 6) or test day 17 (group 7)
Organ / Finding BNT162 c1 BNT162 b2
Group 6: 30 µg/animal Group 7: 100 µg/animal
Males Females Males Females
Perineural tissue of sciatic nerve: 0/10 0/10 10/10** 10/10**
- Inflammation (perineural)
Bone, os femori s with joint (surrounding
tissue):
- Inflammation 0/10 0/10 2/10 9/10**
Ma m mar y gland (Interstitial tissue):
- Inflammation 0/10 4/10 2/10 0/10
Lymph node (ilia c):
6/10*
7/10**
10/10**
10/10**
- Plasmacytosis
- Inflammation 4/10 7/10** 9/10** 6/10*
- Increased cellularity, germinal cente r 10/10 10/10** 10/10 10/10**
Skeletal muscle:
- Infiltration, lymphohi stiogranul ocyt.
0/10
0/10
5/10*
0/10
Spleen:
- Increased haematopoiesis
0/10
0/10
2/10
8/10**
Liver
- Vacuolation, hepatocellular , peripor tal 1/10 10/10** 9/10** 10/10**
…/... number of animals affected per number of animals examined
* signific antly different from control (p ≤ 0.05)
** signific antly different from control (p ≤ 0.01)
Table 35: Incidences of test article -related microscopic findings for the animals treated with
BNT162c1 and BNT162b2
FDA-CBER-2021-5683-1150663
BLA 125742
Table 36: Microscopic findings at terminal sacrifice
Observations: Neo-Plastic and Non Neo-Plastic ---------- --- -- --- -- -- --- -- - MALES -------------- --- -- -- --- -- - ------------- --- -- -- --- -- -- FEMALES -------------- --- -- -- --- -Re moval Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/ Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/
Number of Animals on Study : 15 15 15 15 15 15 15 15 15 15 15 15 15 15
Number of Animals Completed: (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
ADRENAL GLAND, LEFT;
ƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒƒƒ ƒƒƒƒ ƒƒƒ ƒ
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 86.7% 73.3% 66.7% 73.3% 80.0% 86.7% 73.3% 80.0% 80.0% 60.0% 80.0% 93.3% 86.7% 80.0%
Dilation; vascular ...................... 13.3% 26.7% 33.3% 26.7% 20.0% 13.3% 20.0% 20.0% 20.0% 33.3% 20.0% 6.7% 13.3% 20.0%
ADRENAL GLAND, RIGHT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 86.7% 80.0% 80.0% 100.0% 80.0% 86.7% 73.3% 86.7% 86.7% 86.7% 86.7% 86.7% 100.0% 93.3%
Dilation; vascular ...................... 13.3% 20.0% 20.0% 0.0% 20.0% 13.3% 20.0% 13.3% 13.3% 6.7% 13.3% 13.3% 0.0% 6.7%
BONE, OS FEMORIS WITH JOINT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 100.0% 100.0% 100.0% 100.0% 66.7% 100.0% 86.7% 100.0% 93.3% 93.3% 100.0% 53.3% 100.0% 40.0%
Inflammation; mix ed; surrounding tissue . 0.0% 0.0% 0.0% 0.0% 26.7% 0.0% 13.3% 0.0% 6.7% 6.7% 0.0% 40.0% 0.0% 60.0%
BONE MARROW, OS FEMORIS WITH JOINT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 100.0% 33.3% 33.3% 33.3% 33.3% 33.3% 33.3% 100.0% 33.3% 33.3% 33.3% 33.3% 33.3% 33.3%
Increased Cellularity ................... 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7% 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7%
CERVIX;
Examined................................. (-) (-) (-) (-) (-) (-) (-) (15) (15) (15) (15) (15) (15) (14)
Within Normal Limits..................... - - - - - - - 80.0% 53.3% 66.7% 66.7% 73.3% 66.7% 78.6%
Keratinization; epithelial .............. - - - - - - - 20.0% 46.7% 33.3% 33.3% 26.7% 33.3% 21.4%
EPIDIDYMIS, LEFT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (-) (-) (-) (-) (-) (-) (-)
Within Normal Limits..................... 26.7% 13.3% 13.3% 53.3% 33.3% 33.3% 26.7% - - - - - - -
Infiltration, Lymphocytic ............... 73.3% 86.7% 86.7% 46.7% 66.7% 66.7% 73.3% - - - - - - -
EPIDIDYMIS, RIGHT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (-) (-) (-) (-) (-) (-) (-)
Within Normal Limits..................... 33.3% 13.3% 20.0% 33.3% 26.7% 26.7% 13.3% - - - - - - -
Infiltration, Lymphocytic ............... 66.7% 86.7% 80.0% 60.0% 73.3% 73.3% 86.7% - - - - - - -
HEART;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 100.0% 80.0% 86.7% 80.0% 93.3% 100.0% 86.7% 100.0% 86.7% 93.3% 93.3% 93.3% 93.3% 100.0%
Infiltration; lymphohistiocytic ......... 0.0% 6.7% 6.7% 6.7% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0%
Infiltration; mix ed ..................... 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0%
Infiltration, Lymphocytic ............... 0.0% 13.3% 6.7% 6.7% 6.7% 0.0% 13.3% 0.0% 6.7% 6.7% 0.0% 6.7% 6.7% 0.0%
FDA-CBER-2021-5683-1150664
BLA 125742
52 Observations: Neo-Plastic and Non Neo-Plastic ---------- --- -- --- -- -- --- -- - MALES -------------- --- -- -- --- -- - ------------- --- -- -- --- -- -- FEMALES -------------- --- -- -- --- -Re moval Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/ Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/
Number of Animals on Study : 15 15 15 15 15 15 15 15 15 15 15 15 15 15
Number of Animals Completed: (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
INJECTION SITE I;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 60.0% 0.0% 0.0% 0.0% 0.0% 33.3% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 6.7% 6.7%
Fibrosis; intramuscular / interstitial .. 0.0% 66.7% 86.7% 86.7% 86.7% 60.0% 93.3% 0.0% 86.7% 73.3% 93.3% 73.3% 66.7% 93.3%
Fibrosis; inter - / perimuscular ......... 0.0% 100.0% 100.0% 93.3% 100.0% 66.7% 100.0% 0.0% 100.0% 100.0% 100.0% 100.0% 93.3% 93.3%
Inflammation; lymphohistiocytic;
intramuscular / interstitial ...... 0.0% 13.3% 13.3% 33.3% 26.7% 0.0% 20.0% 0.0% 26.7% 6.7% 26.7% 6.7% 0.0% 26.7%
Inflammation; lymphohistiocytic; inter - /
peri mus cul ar ...................... 0.0% 33.3% 26.7% 33.3% 33.3% 6.7% 33.3% 0.0% 26.7% 20.0% 33.3% 26.7% 20.0% 26.7%
Inflammation; mix ed; subcutis ........... 0.0% 66.7% 66.7% 66.7% 66.7% 60.0% 66.7% 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7%
Inflammation; mix ed; intramuscular /
interstitial ...................... 0.0% 60.0% 66.7% 66.7% 60.0% 60.0% 66.7% 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7%
Inflammation; mix ed; inter - /
perimuscular ...................... 0.0% 66.7% 66.7% 66.7% 66.7% 60.0% 66.7% 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7%
Degeneration; myofiber .................. 6.7% 60.0% 60.0% 60.0% 66.7% 53.3% 66.7% 0.0% 60.0% 60.0% 66.7% 66.7% 60.0% 66.7%
Edema; subcutis ......................... 0.0% 40.0% 66.7% 60.0% 53.3% 60.0% 66.7% 0.0% 66.7% 60.0% 66.7% 66.7% 66.7% 66.7%
Edema; intramuscular / interstitial ..... 0.0% 13.3% 46.7% 53.3% 53.3% 60.0% 66.7% 0.0% 66.7% 53.3% 60.0% 66.7% 66.7% 66.7%
Edema; inter - / perimuscular ............ 0.0% 46.7% 66.7% 66.7% 53.3% 60.0% 66.7% 0.0% 66.7% 66.7% 66.7% 66.7% 66.7% 66.7%
Hyperplasia; epidermal .................. 0.0% 60.0% 60.0% 60.0% 66.7% 60.0% 60.0% 0.0% 60.0% 46.7% 53.3% 66.7% 66.7% 66.7%
INJECTION SITE II;
Examined................................. (15) (5) (0) (0) (15) (0) (15) (15) (1) (0) (0) (15) (0) (15)
Within Normal Limits..................... 66.7% 20.0% 0.0% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 0.0% 0.0% 6.7% 0.0% 6.7%
Degeneration; myofiber .................. 0.0% 60.0% 0.0% 0.0% 66.7% 0.0% 66.7% 6.7% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
Regeneration; muscle .................... 0.0% 20.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Hyperplasia; epidermal .................. 0.0% 80.0% 0.0% 0.0% 66.7% 0.0% 46.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 60.0%
Scab; epidermal ......................... 0.0% 40.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Edema; subcutis ......................... 0.0% 60.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
Edema; inter - / perimuscular ............ 0.0% 40.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
Edema; intramuscular / interstitial ..... 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
Necrosis; myofiber ...................... 0.0% 20.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7%
Necrosis; dermis; subcutis .............. 0.0% 20.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Necrosis; traumatic; myofiber ........... 0.0% 20.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Fibrosis; subcutis ...................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Fibrosis; inter - / perimuscular ......... 0.0% 60.0% 0.0% 0.0% 100.0% 0.0% 100.0% 0.0% 100.0% 0.0% 0.0% 93.3% 0.0% 93.3%
Fibrosis; intramuscular / interstitial .. 0.0% 60.0% 0.0% 0.0% 93.3% 0.0% 86.7% 0.0% 0.0% 0.0% 0.0% 80.0% 0.0% 80.0%
FDA-CBER-2021-5683-1150665
BLA 125742
53 Observations: Neo-Plastic and Non Neo-Plastic ---------- --- -- --- -- -- --- -- - MALES -------------- --- -- -- --- -- - ------------- --- -- -- --- -- -- FEMALES -------------- --- -- -- --- -Re moval Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/ Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/
Number of Animals on Study : 15 15 15 15 15 15 15 15 15 15 15 15 15 15
Number of Animals Completed: (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Inflammation; mix ed; intramuscular /
interstitial ...................... 0.0% 80.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
INTESTINE, RECTUM;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 86.7% 80.0% 73.3% 86.7% 60.0% 93.3% 86.7% 80.0% 80.0% 93.3% 60.0% 86.7% 93.3% 46.7%
Infiltration, Eosinophilic; increased ... 0.0% 0.0% 0.0% 13.3% 26.7% 0.0% 0.0% 6.7% 0.0% 0.0% 33.3% 13.3% 0.0% 40.0%
Hyperplasia; mucosa -associated lymphoid
ti ssue ............................ 13.3% 20.0% 26.7% 0.0% 20.0% 6.7% 13.3% 6.7% 20.0% 6.7% 6.7% 6.7% 6.7% 13.3%
KIDNEY, LEFT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0%
Conges ti o n .............................. 93.3% 100.0% 100.0% 100.0% 100.0% 100.0% 93.3% 100.0% 100.0% 100.0% 93.3% 100.0% 100.0% 100.0%
Basophilia; tubule ...................... 13.3% 6.7% 6.7% 13.3% 6.7% 20.0% 13.3% 13.3% 0.0% 0.0% 6.7% 6.7% 0.0% 0.0%
Infiltration, Lymphocytic ............... 26.7% 6.7% 26.7% 20.0% 6.7% 0.0% 20.0% 6.7% 13.3% 6.7% 6.7% 13.3% 6.7% 0.0%
KIDNEY, RIGHT;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Conges ti o n .............................. 93.3% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
Basophilia; tubule ...................... 0.0% 0.0% 6.7% 6.7% 6.7% 13.3% 26.7% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0%
Infiltration, Lymphocytic ............... 6.7% 6.7% 20.0% 6.7% 6.7% 0.0% 6.7% 6.7% 0.0% 0.0% 0.0% 6.7% 6.7% 0.0%
LIVER;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 0.0% 6.7% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Conges ti o n .............................. 100.0% 93.3% 93.3% 100.0% 100.0% 93.3% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0% 100.0%
Hematopoiesis; extramedullary ........... 13.3% 26.7% 13.3% 20.0% 6.7% 6.7% 13.3% 20.0% 20.0% 6.7% 46.7% 33.3% 40.0% 33.3%
Infiltration; mix ed ..................... 6.7% 0.0% 0.0% 0.0% 0.0% 20.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 6.7% 6.7%
Necrosi s .............................. . 6.7% 0.0% 0.0% 13.3% 0.0% 13.3% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Infiltration, Neutrophilic .............. 6.7% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Infiltration, Lymphocytic ............... 60.0% 33.3% 66.7% 53.3% 40.0% 13.3% 33.3% 60.0% 26.7% 46.7% 40.0% 13.3% 33.3% 13.3%
Vacuolation; hepatocellular ............. 6.7% 0.0% 0.0% 13.3% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Vacuolation; hepatocellular; periportal . 6.7% 6.7% 6.7% 0.0% 53.3% 6.7% 60.0% 0.0% 66.7% 40.0% 66.7% 66.7% 73.3% 66.7%
Observations: Neo-Plastic and Non Neo-Plastic ---------- --- -- --- -- -- --- -- - MALES -------------- --- -- -- --- -- - ------------- --- -- -- --- -- -- FEMALES -------------- --- -- -- --- -Re moval Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7: Inflammation; mix ed; subcutis ........... 0.0% 80.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
Inflammation; mix ed; inter - /
peri mus cul ar ...................... 0.0% 80.0% 0.0% 0.0% 66.7% 0.0% 66.7% 0.0% 0.0% 0.0% 0.0% 66.7% 0.0% 66.7%
FDA-CBER-2021-5683-1150666
BLA 125742
54
Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/ Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/
Number of Animals on Study : 15 15 15 15 15 15 15 15 15 15 15 15 15 15
Number of Animals Completed: (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
LUNGS WITH BRONCHI; (continued)
Hemorrhage; acute ....................... 26.7% 20.0% 33.3% 33.3% 20.0% 26.7% 33.3% 6.7% 13.3% 13.3% 6.7% 6.7% 6.7% 0.0%
Hyperplasia; bronchial -associated lymphoid tissue ................... 46.7% 60.0% 33.3% 60.0% 40.0% 40.0% 20.0% 13.3% 26.7% 26.7% 46.7% 26.7% 33.3% 33.3%
Infiltration, Eosinophilic; perivascular 20.0% 6.7% 6.7% 13.3% 20.0% 6.7% 6.7% 6.7% 0.0% 13.3% 26.7% 13.3% 0.0% 53.3%
LYMPH NODE, CERVICAL;
Examined................................. (13) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (14)
Within Normal Limits..................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Hi stocyt o si s ............................ 100.0% 100.0% 93.3% 86.7% 100.0% 93.3% 86.7% 93.3% 86.7% 100.0% 93.3% 100.0% 86.7% 92.9%
E r yt h r opha gocytos i s ..................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0%
Pigmentation; brown; macrophage ......... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 20.0% 6.7% 0.0%
Hemorr ha g e .......................... ... 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Pl asmac yt o si s ........................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Increased Cellularity; germinal center .. 100.0% 100.0% 100.0% 93.3% 100.0% 100.0% 93.3% 86.7% 100.0% 100.0% 93.3% 100.0% 100.0% 85.7%
LYMPH NODE, ILIAC;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (14) (15) (14)
Within Normal Limits..................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Hi stocyt o si s ............................ 100.0% 93.3% 100.0% 93.3% 100.0% 93.3% 93.3% 93.3% 93.3% 73.3% 66.7% 100.0% 100.0% 92.9%
Pl asmac yt o si s ........................... 0.0% 33.3% 46.7% 73.3% 73.3% 40.0% 73.3% 0.0% 40.0% 66.7% 73.3% 100.0% 53.3% 100.0%
Infiltration, Eosinophilic .............. 6.7% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 7.1% 0.0% 0.0%
Hemorrhage; acute ....................... 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0%
Inflammati o n ............................ 0.0% 33.3% 0.0% 0.0% 33.3% 26.7% 60.0% 0.0% 40.0% 20.0% 0.0% 50.0% 46.7% 42.9%
Infiltration; macrophage ................ 0.0% 0.0% 6.7% 6.7% 33.3% 20.0% 33.3% 0.0% 20.0% 0.0% 20.0% 35.7% 33.3% 28.6%
Increased Cellularity; germinal center .. 86.7% 93.3% 93.3% 100.0% 100.0% 100.0% 100.0% 46.7% 86.7% 100.0% 86.7% 100.0% 93.3% 100.0%
NERVE, SCIATIC;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (14) (15)
Within Normal Limits..................... 100.0% 80.0% 100.0% 93.3% 53.3% 100.0% 20.0% 100.0% 100.0% 86.7% 73.3% 26.7% 100.0% 26.7%
Inflammation; perineural ................ 0.0% 20.0% 0.0% 6.7% 46.7% 0.0% 80.0% 0.0% 0.0% 6.7% 26.7% 73.3% 0.0% 73.3%
Vacuolati o n ............................. 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0%
PROSTATE GLAND;
Examined................................. (15) (14) (15) (15) (14) (15) (15) (-) (-) (-) (-) (-) (-) (-)
Within Normal Limits..................... 80.0% 85.7% 66.7% 60.0% 85.7% 73.3% 86.7% - - - - - - -
Infiltration; mix ed ..................... 0.0% 7.1% 6.7% 6.7% 0.0% 0.0% 0.0% - - - - - - -
Inflammation; purulent .................. 6.7% 0.0% 0.0% 6.7% 7.1% 13.3% 0.0% - - - - - - -
Infiltration, Lymphocytic ............... 13.3% 7.1% 26.7% 26.7% 7.1% 13.3% 13.3% - - - - - - -
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55 Observations: Neo-Plastic and Non Neo-Plastic ---------- --- -- --- -- -- --- -- - MALES -------------- --- -- -- --- -- - ------------- --- -- -- --- -- -- FEMALES -------------- --- -- -- --- -Re moval Reasons: All of those SELECTED Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Group 1: Group 2: Group 3: Group 4: Group 5: Group 6: Group 7:
Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/ Control 30 µg/ 10 µg/ 30 µg/ 100 µg/ 30 µg/ 100 µg/
Number of Animals on Study : 15 15 15 15 15 15 15 15 15 15 15 15 15 15
Number of Animals Completed: (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
SPLEEN;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 20.0% 6.7% 53.3% 26.7% 33.3% 53.3% 53.3% 20.0% 20.0% 46.7% 20.0% 40.0% 20.0% 13.3%
Conges ti o n .............................. 80.0% 93.3% 40.0% 73.3% 53.3% 46.7% 40.0% 80.0% 80.0% 46.7% 80.0% 40.0% 80.0% 66.7%
Hematopoiesis; increased ................ 0.0% 0.0% 20.0% 0.0% 13.3% 0.0% 13.3% 0.0% 0.0% 13.3% 0.0% 46.7% 0.0% 53.3%
STOMACH, GLANDULAR;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 6.7% 0.0% 33.3% 13.3% 6.7% 33.3% 0.0% 6.7% 20.0% 40.0% 0.0% 0.0% 46.7% 20.0%
Infiltration, Eosinophilic .............. 93.3% 93.3% 60.0% 86.7% 93.3% 60.0% 93.3% 93.3% 66.7% 53.3% 100.0% 100.0% 46.7% 73.3%
Infiltration, Lymphocytic ............... 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0% 0.0% 0.0% 0.0%
Dilation; glandular ..................... 0.0% 13.3% 13.3% 6.7% 6.7% 6.7% 6.7% 0.0% 6.7% 13.3% 6.7% 6.7% 6.7% 13.3%
THYMUS;
Examined................................. (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... 66.7% 46.7% 46.7% 60.0% 60.0% 46.7% 53.3% 46.7% 33.3% 80.0% 60.0% 66.7% 53.3% 40.0%
Cyst ............................ ........ 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 6.7% 0.0%
Hemorrhage; acute ....................... 33.3% 53.3% 53.3% 40.0% 40.0% 53.3% 46.7% 53.3% 66.7% 20.0% 40.0% 33.3% 40.0% 60.0%
UTERUS;
Examined................................. (-) (-) (-) (-) (-) (-) (-) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... - - - - - - - 100.0% 80.0% 66.7% 86.7% 93.3% 100.0% 93.3%
Dilatio n ............................. ... - - - - - - - 0.0% 20.0% 33.3% 13.3% 6.7% 0.0% 6.7%
VAGINA;
Examined................................. (-) (-) (-) (-) (-) (-) (-) (15) (15) (15) (15) (15) (15) (15)
Within Normal Limits..................... - - - - - - - 73.3% 46.7% 53.3% 66.7% 60.0% 60.0% 60.0%
Keratinization; epithelial .............. - - - - - - - 26.7% 53.3% 46.7% 33.3% 40.0% 40.0% 40.0%
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Recovery sacrifice
At the end of the recovery period (day 31 for group 6 and day 38 for all other groups), most of
the microscopic findings reported at the injection sites, iliac lymph node, surrounding tissue of
the injection sites (surrounding tissue of bone, os femoris wi th joint; perineural tissue of sciatic
nerve; interstitial tissue of mammary gland; skeletal muscle) and spleen were partially or
completely recovered in all animals.
Some inflammatory lesions were still reported at the injection sites and the surrounding tissues in
some animals. These lesions were less severe (minimal to mild).
The infiltration of macrophages in the iliac lymph nodes of recovery animals were regarded as a
consequence of phagocytosis relating to the inflammatory reactions at the injection sites. Test
article -related minimal to mild increases in the cellularity of bone marrow and extramedullary
hematopoiesis in the spleen was fully recovered at the end of recovery phase.
Test article -related vacuolation of hepatocytes in the portal regions of the liver was fully
recovered at the end of recovery phase. The incidence and the severity of the remaining findings
were markedly reduced when compared to the main study animals.
Discussion synopsis
Inflammation was generally most at the end of dosing in groups 5 and 7. This is followed by 30
μg BNT162a1/animal group. Ulceration at the injection site was present only in rats administered BNT162a1. The inflammation was partially or fully resolved at the end of the recovery phase.
Increased cellularit y of the germinal centers of the draining (iliac) lymph node and plasmacytosis
were reported. This is consistent with the anticipated immune activation by the test articles. Increases in bone marrow cellularity (increased hematopoiesis) and extramedullary
hematopoiesis in the spleen were reported. This is consistent with a response to inflammation
and immune responses induced by the test article.
Test article -related vacuolation of portal hepatocytes was reported in all groups. The vacuolation
was unassoci ated with markers of hepatocyte damage (i.e. ALAT, ASAT) and has been reported
in animals administered pegylated compounds only. This finding was fully reversed at the end of the recovery phase.
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57 Body temperature:
No test article -related effects on body t emperature were reported.
Figure 12: Body temperature of male rats treated once weekly, mean values per group
Figure 13: Body temperature of female rats treated once weekly, mean values per group
Serology:
In this study the immunogenicity of the administered SARS -CoV-2-S protein targeted RNA
vaccines BNT162a1, BNT162b1, BNT162b2, and BNT162c1 was investigated. At study day 10, serum samples were collected from animals treated with BNT162c1 (group 6). At stud y day 17
serum samples were collected from animals treated with BNT162a1, BNT162b1, and BNT162b2
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58 (groups 2, 3, 4, 5, and 7). Antibody immune response analyzed by S1 domain and RBD sub-
domain specific ELISA as well as VSV/SARS -CoV-2-S-based pseudovirus neut ralization assay
(pVNT).
All BNT162 vaccine candidates elicited a SARSCoV - 2-S protein specific antibody response
directed against the S1 domain and the RBD sub-domain. Antibody responses translated into neutralizing activity as reported in the VSV/SARS -CoV -2-S pseudovirus neutralization test.
BNT162 vaccine candidates showing higher antigen-specific antibody titers also displayed more pronounced virus neutralization effect.
Figure 14: Antibody titer resulting in 50% pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in 50% pseudovirus neutralization (pVN50) are shown by dots; group mean values are indicated by horizontal bars (±SEM, standard error of the mean).
Figure 15: antibo dy titer resulting in 90% pseudovirus neutralization activity (pVN50).
Individual VNT titers resulting in 90% pseudovirus neutralization (pVN90) are shown by dots; group mean values are indicated by horizontal bars (±SEM, standard error of the mean).
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59 Test article related effects Effects considered incidental
↓ Triglycerides
↑ Gamma -GT
↓ Reticulocytes
↓ Platelet
↑ Monocytes
↑ Neutrophils
↑ Eosinophils
↑ Basophils
↑ WBC
↑ LUC
↑ Fibrinogens
↓ PCT%
↑ Alpha1-acid glycoproteins
↑ Alpha2-macroglobulins
↑ Epididymides weight
↑ Mesenteric lymph nodes weight
↑ Spleen weight
↑ Thyroid weight for females
Injection site findings (indurated, incrusted, and
thickened skin)
Enlarged iliac lymph nodes
Enlarged spleen
↑ Cellularity of bone marrow
Immune responses in groups 2, 3, 4, 5, and 7 ↓ Thymus weight
IFN-gamma, TNF -alpha, IL -1beta,
and IL -10
Table 37: Test article related effects
Assessment:
No treatment -related, mortality, nor any toxicologically relevant changes in clinical signs, food
consumption, body temperature, ophthalmic changes, urinalysis, or auditory examination were
reported.
A triglyceride is an ester derived from glycerol and three fatty acids .4 Triglycerides are the main
constituents of body fat in humans and animals, as well as vegetable fat .5 They are also present
in the blood to enable the bidirectional transference of adipose fat and blood glucose from the
liver, and are a major component of human skin oils .6 In the human body, high levels of
triglycerides in the bloodstream have been linked to atherosclerosis and, by extension, the risk of
4 "Nomenclature of Lipids" . IUPAC -IUB Commission on Biochemical Nomenclature (CBN). Retrieved
2007- 03-08.
5 Nelson, D. L.; Cox, M. M. (2000). Lehninger, Principles of Biochemistry (3rd ed.). New York: Worth
Publishing. ISBN 1-57259- 153-6 .
6 Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, M.
(1983). "Human stratum corneum lipids: characterization and regional variations". J. Lipid Res. 24: 120–
130. PMID 6833889
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60 heart disease7 and stroke .8 The decrease in triglyceride levels were not considered of any
toxicological importance.
Gamma -glutamyl transferase (GGT) is a membrane -bound enzyme catabolizing reduced
glutathione to cysteine and glycine in Meist er's γ-glutamyl cycle ( Orlowski and Meister, 1970 ).9
This delivers cysteine for intracellular synthesis of glutathione, the major thiol anti-oxidant.
Elevated serum levels of GGT are markers of oxidative stress, resulting from factors including
alcohol, heavy metals, cardiovascular disease and diabetes. Furthermore, higher serum levels of GGT, within the normal range, are associated with an increased cancer risk. High levels of GGT
seem to increase the risk of progression of high-grade cervical dysplasia to invasive carcinoma.
10
Reticulocytes are immature red blood cells (RBCs). In the process o f erythropoiesis (red blood
cell formation), reticulocytes develop and mature in the bone marrow and then circulate for
about a day in the blood stream before developing into mature red blood cells. Like mature red
blood cells, in mammals, reticulocytes do not have a cell nucleus .
11 Abnormally low numbers of
reticulocytes can be attributed to chemotherapy , aplastic anemia , pernicious anemia , bone
marrow malignancies, problems of erythropoietin production, various vitamin or mineral
deficiencies ( iron, vitamin B 12, folic acid ), disease states ( anemia of chronic disease ) and other
causes of anemia due to poor RBC production.12
The cells that circulate within our blood and bind together when they recognize damaged blood
vessels are called platelets . The platelets bind to the site of the damaged vessel in any cut,
thereby causing a blood clot to stop bleeding. Platelets are literally shaped like small plates in their non-active form. A damaged blood vessel will send out a signal and when platelets receive that signal, they’ll respond by traveling to that area and transform into their “active” formation.
To make contact with the broken blood vessel, platelets grow long tentacles and then resemble a
spider or an octopus. A normal platelet count ranges from 150,000 to 450,000 platelets per microliter of blood. Having more than 450,000 platelets is a condition called thrombocytosis ;
having less than 150,000 is known as thrombocytopenia. A decrease in platelet levels is called
thrombocytopenia. Easy bruising, and frequent bleeding from the gums, nose, or GI tract are the
symptoms of thrombocytopenia. Thrombocytopenia happens when something is preventing your
body from producing platelets . There are a wide range of causes, including: medications, an
inherited condition, certain types of cancer (such as leukemia or lymphoma), chemotherapy
treatment for cancer, kidney infection or dysfunction, or too much alcohol.
13
7 "Boston scientists say triglycerides play key role in heart health" . The Boston Globe. Retrieved 2014- 06-
18.
8 Drummond, K. E.; Brefere, L. M. (2014). Nutrition for Foodservice and Culinary Professionals (8th ed.).
John Wiley & Sons. ISBN 978-0- 470-05242-6 .
9 Orlowski M, Meister A. The γ -gluta myl cycle: a possible transport system for a mino acid s. PNAS. 1970;67 :1248 –
1255.
10 https://www ncbi nlm.nih.gov/pmc/articles/PMC3341856/
11 https://en.wikipedia.org/wiki/Reticulocyte
12 https://www.uofmhealth.org/health -library/hw203366
13https://www.hopkinsmedicine.org/heart_vascular_institute/centers_excellence/women_cardiovascular_health_cent
er/pa tient_information/health_topics/platelets html
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61 Monocytosis could be indicative of the intended immune response or could be secondary to
muscle damage at the site of injection as an indication of inflammation and repair. The increases
in the monocyte count might be related to test article treatment.
Neutrophils are key components in the system of defense against infection. An individual with
absence or scarcity of neutrophils (neutropenia) is vulnerable to infection. The increase in neutrophils might be related to the immune responses initiated by the test article treatment.
Eosinophils are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. They are granulocytes that develop during hematopoiesis in the bone marrow before migrating into blood.
Basophils play a role in both parasitic infections and allergies. Basopenia has been reported in association with autoimmune urticaria.
White blood cells (WBCs ) (also called leukocytes or leucocytes) are the cells of the immune
system that are involved in protecting the body against both infectious disease and foreign
invaders. All white blood cells are produced and derived from multipotent cells in the bone
marrow known as hematopoietic stem cells . Leukocytes are found throughout the body,
including the blood and lymphatic system .
14 The increase in WBC might be related to the
immune response induced by the test art icle treatment.
LUC is a measurement of the large, peroxidase -negative cells which cannot be further
characterized (i.e. as large lymphocytes, virocytes, or stem cells) present in a biological specimen. In LUC are found large lymphoid cells , more immatur e lymphocytes and other cells.
If the value is higher than normal, blood counts should be checked under a microscope slide.
The increases in fibrinogen levels were not considered frank toxicity but rather an anticipated
effect associated with an immunolog ical response.
Relative volume of thrombocytes ( very large cells in the bone marrow called
megakaryocytes )/Plateletcrit (measure of total platelet mass) percent (PCT%) was decreased in
groups 3, 5, and 7 males and females at study day 17. This is crucial to normal blood clotting. Alpha -1-acid glycoprotein ( α
1AGp,[1] AGP or AAG ), which is modulated by two polymorphic
genes , is an acute phase ( acute phase protein ) plasma alpha -globulin glycoprotein . It has a
normal plasma concentration between 0.6 -1.2 mg/mL (1 -3% plasma protein) and is synthesized
primarily in hepatocytes (5). Plas ma levels are affected by pregnancy, burns, certain drugs, and
certain diseases, particularly HIV (5). The function of alpha -1-acid glycoprotein is to act as a
carrier of basic and neutrally charged lipophilic compounds. It is known as the primary carrier of basic (positively charged) drugs (whereas albumin carrie s acidic (negatively charged) and neutral
drugs), steroids , and protea se inhibitors (5, 6). AGP shows a complex interaction with thyroid
homeostasis. Alpha -1-acid glycoprotein (in low concentrations) was reported to stimulate the
14 Maton, D., Hopkins, J., McLaughlin, Ch. W., Johnson, S., Warner, M. Q., LaHart, D., & Wright, J. D., Deep V.
Kulkarni (1997). Human Biology and Health. Englewood Cliffs, New Jersey, US: Prentice Hall. ISBN 0-13-9 81176 -
1.
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62 thyrotropin (TSH) receptor and intracellular accumulation of cyclic AMP . However, high AGP
concentrations inhibited TSH signaling (7, 8). Alpha -1-acid glycoprotein has been identified as
one of four potentially useful circulating biomarkers for estimating the five -year risk of all -cause
mortality (the other three are albumin , very low -density lipoprotein particle size, and citrate ) (9).
Alpha -1-acid glycoprotein increases in obstructive jaundices while diminishes in hepatocellular
jaundice and in intestinal infections .15
Alpha -2-macroglobulin (α2M) is a large plasma protein found in the blood, mainly produced by
the liver, and also locally synthesized by macrophages , fibroblasts , and adrenocortical cells . It
acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions
as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein and as an inhibitor of
coagulation by inhibiting thrombin. Because it also binds to numerous growth factors and cytokines, such as platelet -derived growth factor, basic fibroblast growth factor, TGF -β, insulin,
and IL -1β, it may act as a carrier protein. In the nephrotic syndrome when other lower molecular
weight proteins are lost in the urine, the concentration of alpha -2-macroglobulin rises 10 -fold or
more
16.
The epididymis is a tube that connects a testicle to a vas de ferens in the male reproductive
system . It is present in all male reptiles, birds, and mammals. It is a single, narrow, tightly -coiled
tube connecting the efferent ducts from the rear of each testicle to its vas deferens. An
inflammation of the epididymis is called epididymitis . It is much more common than testicular
inflammation, termed orchitis .
17
The increases in the weights of mesenteric lymph nodes and the enlargement of the iliac lymph
nodes might be related to the immune response due to test article treatment.
The external iliac lymph nodes are eight to ten in number, that lie along the external iliac vessels .
They are arranged in three groups, one on the lateral, another on the medial, and a third on the
anteri or aspect of the vessels; the third group is, however, sometimes absent. Their principal
afferents are derived from the inguinal lymph nodes , the deep lymphatics of the abdominal wall
below the umbilicus and of the adductor region of the thigh, and the lymphatics from the glans
penis, glans clitoris , the membranous urethra , the prostate , the fundus of the urinary bladder , the
cervix uteri , and upper part of the vagina
18.
Spleen weight increase might be related to the intended immune response. The spleen plays
important roles in regard to red blood cells and the immune system19. It removes old red blood
cells and holds a reserve of blood in case of hemorrhagic shock while also recycling iron. As a part of the mononuclear phagocyte system, it me tabolizes hemoglobin removed from senescent
erythrocytes. The globin portion of hemoglobin is degraded to its constitutive amino acids, and the heme portion is metabolized to bilirubin, which is subsequently shuttled to the liver for
15 https://en.wikipedia.org/wiki/Orosomucoid
16 https://en.wikipedia.org/wiki/Alpha -2-Macroglobulin
17 https://en.wikipedia.org/wiki/Epididymis
18 https://en.wikipedia.org/wiki/External ilia c lymph nodes
19 Spleen, Internet Encyclopedia of Science.
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63 removal20. It synthesizes antibodies in its white pulp and removes antibody-coated bacteria along
with antibody-coated blood cells by way of blood and lymph node circulation.
The thyroid gland controls how quickly the body makes proteins and uses energy. And, controls
how sensitive the body is to other hormones . It produces the thyroid hormones [ triiodothyronine
(T3) and thyroxine (sometimes referred to as tetraiodothyronine (T 4)]. These hormones regulate
the growth and rate of function of many other systems in the body. T 3 and T 4 are synthesized
from iodine and tyrosine . The thyroid also produces calcitonin , which plays a role in calcium
homeostasis . Hormonal output from the thyroid is regulated by thyroid -stimulating hormone
(TSH) produced by the anterior pituitary . TSH is regulated by thyrotropin-releasing hormone
(TRH) produced by the hypothalamus .
Test article -related injection site findings (indurated, incrusted, and thickened skin) were
reported. Inflammation is a relatively common occurrence as part of the acute phase response following administration of some vaccines.
In all test article -treated groups, minimal to mild increases in the cellularity of bone marrow we re
reported. They were likely secondary to inflammation-related platelet activation and consumption.
Test article -related immune responses in groups 2, 3, 4, 5, and 7 were reported.
The thymus is a specialized primary lymphoid organ of the immune system . Within the thymus,
T cells or T lymphocytes mature. T cells are critical to the adaptive immune system , where t he
body adapts specifically to foreign invaders. The thymus is composed of two identical lobes and
is located anatomically in the anterior superior mediastinum , in front of the heart and behind the
sternum .
21 One of the major characteristics of vertebrate immunology is thymic involution , the
shrinking of the thymus with age, resulting in changes in the architecture of the thymus and a
decrease in tissue mass.22 T-cells are named for the thymus where T-lymphocytes migrate from
the bone marrow to mature. Its regression has been linked to the reduction in immunosurveillance in the elderly.
23
No clear important changes in the levels of cytokines (IFN -gamma, TNF -alpha, IL -1beta, and
IL-10) were reported.
Adverse gross alteration that could be indicative of systemic or local toxicity was not reported.
Based on the overall findings in this study, it can be concluded that in Wistar rats, repeat dose on study days 1, 8, and 15 had no adverse effects in terms of systemic toxicity at the dose level of
20 Mebius RE, Kra al G. (2005). Structure and function of the spleen. Na t Rev Immunol. 5(8):606 -16.
21 https://en.wikipedia.org/wiki/Thymus.
22 Shanley D.P.; Danielle A.W.; Manley N.R.; Palmer D.B.; et al. (2009). "An evolutionary perspective on the
mechanisms of immu nosenescence". Trends Immunol. 30 (7): 374 –381. doi:10.1016/j.it.2009.05.001 .
PMID 19541538
23 Linton P.J.; Dorshkind K. (2004). "Age -related changes in lymphocyte development and function". Nat. Immu nol.
5 (2): 133 –139. doi:10.1038/ni1033 . PMID 14749784
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64 10, 30, or 100 µg/animal. However, due to the significant decrease in the reticulocyte levels,
hematology results should be closely monitored during any clinical trial.
GLP study deviations or amendments: Deviations or amendments were not included in this
study submission and expected to be included in the final study report.
Investigators Brochure: Having read and evaluated the Investigators Brochure, is it a fair,
objective and reasonable summary of the toxicology data – yes (X) or no ().
Internal Communication:
Due to the significant decreases in the platelet’s and reticulocyte’s levels, close m onitoring to the
hematology data in any clinical trial is highly recommended. Conclusions:
Based on nonclinical toxicity assessments, there are no significant safety issues to preclude the
IND from going into effect
Study number 2:
Title and study number: 17-day intramuscular toxicity study of BNT162B2 (V9) and
BNT162B3C In Wistar Han rats with a 3-week recovery. Study number: 20GR142.
Performing laboratory: Pfizer Worldwide Research & Development Drug Safety Research & Development Eastern Point Road Groton, CT 06340 USA.
Study initiation date: June 23, 2020
Final report date: August 13, 2020 Test article batch/lot:
Test Article Lot Number Expira tion Da te
BNT162b2 (V9) COVVAC/270320 27 Sep 2020
BNT162b3c BCV/040620 04 Dec 2020
0.9% sterile sa line J8L247 31 Mar 2021
Animal species and strain: Rat/Wistar/Crl:WI(Han)
Breeder/s upplier : Charles River Laboratories Raleigh, NC
Number of animal per group and sex: 15/sex/group
Age: 9 weeks
Body weight range:
Males: 243.1 grams - 291.6 grams
Females: 172.9 grams - 209.5 grams
Route and site of administration: Intramuscular (IM)
Volum e of injection: 60 µL
Frequency of administration and study duration:
Animals were treated on study days 1, 8, and 15 into the left hindlimb quadriceps muscle
Dose : See study design
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65 Stability: Analysis of stability, homogeneity and concentration of the test article under test
conditions was not performed as part of the study. Stability studies were performed by the
sponsor of the IND. At the time of submitting this study, stability studies with the first clinical
trial material batch have just been started. Up to now no results are available. Stability data will be included in any upcoming amendment. The table below shows the protocol of stability study I
for CTM drug substance batches:
Table o f protocol of stability study I for CTM drug substance batches at different storage
conditions
condi
tions
Stability of was reported.
Means of administration: Intramuscular (IM)
Report status: Final report
Experimental design:
Animals were randomized and assigned to 3 different groups. Each group consisted of 15/sex/group. The first 10 animals/sex/group, by ascending animal order, were designated for necropsy at the end of the dosing phase. The remaining 5 a nimals were retained for the recovery
phase. Animals were dosed by IM on study days 1, 8, and 15. The details of the study design are
listed in the following table:
Table of experimental design
Group
Number Test Article or Vehicle
Dose (µg RNA/Dose Day) Dose Volume
(µL/injection site)a
Animal Numbers
Males Females
1 0b 60 1-15 46-60
2 30c 60 16-30 61-75
3 30d 60 31-45 76-90
a. Each animal received a single intramuscular injection on each dose day.
(b) (4)
(b) (4)
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BLA 125742
66 b. Sterile sa line.
c. BNT162b2 (V9).
d. BNT162b3c.
Methods:
Randomization procedure: Yes
Statistical analysis plan: Yes.
The following parameters were evaluated:
General (Cageside) Clinical
Observations: Days of Study Time Points
Prior to the Initiation of Dosing
(PID) Once da ily
Non-dosing Days (Dosing Phase) Twice da ily, except on days when d e ta iled
clinica l observations were performed, then
only once daily
Dosing Days (Dosing Phase) Pre dose, except on days that pre dose
deta iled clinical observations were
performed, 4 hours after the la st animal
was dosed, a nd at the end of the workday.
On 06 Jul 2020 (day 1), clinica l signs
were not conducted at the end of the
workday for Animals 001-090.
Recovery Phase Days Twice da ily
Deta iled Clinical
Observations: Deta iled clinical observations were performed twice prior to the initia tion of
dosing, twice weekly at approximately the same time body weights were
performed, and on the day(s) of necropsy.
Body Weight: All a nimals were weighed twice prior to the initia tion of dosing on PID Phase
days 1 and 6, pre dose on dosing phase days 1, 8, a nd 15; on dosing phase
days 4 and 11 (non-dosing), a nd a fasted weight was collected just prior to
scheduled necropsy. Body weights were collected on recovery phase days 1,
4, 8, 11, 15, 18, a nd 21.
Food Consumption: Qua ntitative food consumption was recorded on dosing phase days 4, 8, 11,
and 15 a nd on recovery phase days 4, 8, 11, 15, 18, and 21.
Ophthalmology: Ophthalmic examinations were performed once prior to the initia tion of dosing
(following ra ndomization) on PID phase days 7/8 (males/females) a nd on
dosing phase days 15/16 (males/females).
Recovery animals were not examined at the end of the recovery phase.
See the ophthalmology report in Appendix B for complete materials a nd
methods.
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67 General (Cageside) Clinical
Observations: Days of Study Time Points
Prior to the Initiation of Dosing
(PID) Once da ily
Non-dosing Days (Dosing Phase) Twice da ily, except on days when d e ta iled
clinica l observations were performed, then
only once daily
Dosing Days (Dosing Phase) Pre dose, except on days that pre dose
deta iled clinical observations were
performed, 4 hours after the la st animal
was dosed, a nd at the end of the workday.
On 06 Jul 2020 (day 1), clinica l signs
were not conducted at the end of the
workday for Animals 001-090.
Recovery Phase Days Twice da ily
Injection Site Scoring
(Dermal Assessment): Injection sites were observed during the dosing phase once pre dose and
approximately 4 a nd 24 hours post dose on a ll a nimals. Animals with a score
of 2 or greater at 24 hours post dose had additional evaluations at 48- and 72 -
hours post-dose. Animals with a continued score of 2 or grea ter at 72 hours
post-dose had additional evaluations at 120 and 144 hours post-dose. After
dosing on day 15, a 72-hour post dose evaluation was conducted on recovery
a nimals only. Injection site score was recorded according to a standardized
ra ting sca le (Draize, 1959)24.
On dosing phase day 1 (06 Jul 2020), pre dose dermal assessments were
collected on a ll a nimals for right-side injection sites (non-injection site), and
at 4 hours post dose, dermal a ssessments were collected on animals 1-7, 9
(group 1, m a les), a nd 46-58 (group 1, females) for right-side injection sites
(non -injection site).
Body Temperature: Body temperature was collected on all a nimals once prior to the initia tion of
dosing on PID phase day 6, pre-dose on dosing phase days 1, 8, a nd 15, a nd
at a pproximately 4- and 24 -hours post-dose from a ll a nimals.
Table 39: parameters evaluated
Clinical l aboratory measurements
Schedule for Collection of Sa mples for Clinical Laboratory Measurements
Parameter Day of Study
Dosing Phase Recovery Phase
Da y
4 Da y
17e Da y
22
Hematology Xa,c Xc Xc
Coa gulation NA Xc Xc
Clinica l Chemistry
(Core Chemistry) Xb,c Xc Xc
Clinica l Chemistry
(Other Biomarkers – Acute
Phase Proteins)/Serumd Xb,c Xc Xc
Urina lysis NA X X
NA = Not a pplicable; X = Scheduled collection.
24 Draize JH. 1959 (2nd printing 1965). Appraisal of the Safety of Chemicals in Foods, Drugs a nd Cosmetics.
Dermal Toxicity, pp. 46 -59. Published by: The Association of Food and Drug Officials of the United States, Topeka,
Kansas.
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68 a. First 7 a nimals/sex/group.
b. Last 8 animals/sex/group.
c. Blood sa mples were collected from animals in a fasted state, with the exception of same day redraws.
d. Assay performed using shared clinical chemistry sample.
e. Eva luated on animals scheduled for necropsy.
Table 40: Clinical laboratory measurements
Antibody (Serology) response to vaccine components
Sample Collection and Storage Conditions
Groups: 1-3
Collection Intervals: PID Phase Day 8 and Dosing Phase Day 17a, and Recovery Phase Day 21a
Collection Time Points: PID Phase Day 8, Dosing Phase Day 17, and Recovery Phase Day 21: Once
Anima ls/Time Point: All a nimals
Anticoa gulant: No Anticoagulant
Collection Volume per
Sa mple: PID Phase Day 8: Approximately 0.7 mL
Dosing Phase Day 17 and Recovery Phase Day 21: Approximately 1 mL
Sa mple Processing: Sa mples were processed and stored as appropriate within 2 hours of
collection
Sa mple Storage Conditions: Approximately -60ºC or lower
PID = Prior to initia tion of dosing.
a. Sa mples collected prior to necropsy.
Table 41: Antibody (Serology) response to vaccine components
Postmortem procedures:
Animals (10/sex/group) were euthanized on dosing phase day 17 (2 days after the last dose).
Remaining animals were euthanized on recovery phase day 22.
Necropsy, tissue collection, organ weights, macroscopic tissue evaluation, and microscopic examination were performed. Bone marrow smears were collected from all animals.
Tissues Collected Organs
Weighed
(All Dose
Groups) Tissues Processed for Slide Preparation (X)
Dose Group
Group 1 Group 2 Group 3
Artery, Aorta X X X
Bone Marrow, Sternum X X X
Bone, Sternum X X X
Bra in X X X X
Cervix X X X
Epididymis X X X X
Esophagus X X X
Eye X X X
Gland, Adrenal X X X X
Gland, Harderian X X X
Gland, Lacrimal
(Extra orbital) X X X
Gland, Mammary X X X
Gland, Parathyroid X X X
Gland, Pituitary X X X
Gland, Prostate X X X X
Gland, Salivary X X X
Gland, Seminal Vesicle X X X
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69 Tissues Collected Organs
Weighed
(All Dose
Groups) Tissues Processed for Slide Preparation (X)
Dose Group
Group 1 Group 2 Group 3
Gland, Thyroid X X X
Gut-Associated Lymphoid
Tissue X X X
Heart X X X X
Joint X X X
Kidney X X X X
La rge Intestine, Cecum X X X
La rge Intestine, Colon X X X
La rynx
Liver X X X X
Lung X X X
Lymph Node, Draining X X X
Lymph Node, Inguinal X X X
Lymph Node, Mesenteric X X X
Macroscopic Findings X X X
Muscle, Skeletal X X X
Nerve, Optic X X X
Nerve, Peripheral X X X
Ovary X X X X
Oviduct X X X
Pancreas X X X
Site, Injection X X X
Skin X X X
Sma ll Intestine,
Duodenum X X X
Sma ll Intestine, Ileum X X X
Sma ll Intestine, Jejunum X X X
Spinal Cord X X X
Spleen X X X X
Stom ach X X X
Testis X X X X
Thymus X X X X
Tongue X X X
Tra chea X X X
Ureter X X X
Urinary Bladder X X X
Uterus X X X
Va gina X X X
Table 42: Tissue collection, organ weights and tissues processed for slide preparation – Dosing
phase
Tissues Collected Organs
Weighed
(All Dose
Groups) Tissues Processed for Slide Preparation (X)
Dose Group
Group 1 Group 2 Group 3
Artery, Aorta
Bone Marrow, Sternum X X X
Bone, Sternum
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70 Tissues Collected Organs
Weighed
(All Dose
Groups) Tissues Processed for Slide Preparation (X)
Dose Group
Group 1 Group 2 Group 3
Bra in X
Cervix
Epididymis X
Esophagus
Eye
Gland, Adrenal X
Gland, Harderian
Gland, Lacrimal
(Extra orbital)
Gland, Mammary
Gland, Parathyroid
Gland, Pituitary
Gland, Prostate X
Gland, Salivary
Gland, Seminal Vesicle
Gland, Thyroid
Gut-Associated Lymphoid
Tissue
Heart X
Joint X X X
Kidney X
La rge Intestine, Cecum
La rge Intestine, Colon
La rynx
Liver X X X X
Lung
Lymph Node, Draining X X X
Lymph Node, Inguinal X X X
Lymph Node, Mesenteric
Macroscopic Findings X X X
Muscle, Skeletal X X X
Nerve, Optic
Nerve, Peripheral
Ovary X
Oviduct
Pancreas
Site, Injection X X X
Skin
Sma ll Intestine,
Duodenum
Sma ll Intestine, Ileum
Sma ll Intestine, Jejunum
Spinal Cord
Spleen X X X X
Stom ach
Testis X
Thymus X
Tongue
Tra chea
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71 Tissues Collected Organs
Weighed
(All Dose
Groups) Tissues Processed for Slide Preparation (X)
Dose Group
Group 1 Group 2 Group 3
Ureter
Urinary Bla dder
Uterus
Va gina
Table 43: Tissue collection, organ weights and tissues processed for slide preparation – Recovery
phase
Results:
No test article -related mortality was reported.
Clinical chemistry and hematology:
Clinical chemistry
CLINICAL CHEMISTRY
MEASUREMENT RELATED
TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT STUDY DAY
(SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
than 1.5 so indicated otherwise ≥ 1.5) ) NOT OF NOTE
ELECTROLYTE BALANCE Ca lcium, chloride, potassium, sodium,
phosphorus
CARBOHYDRATE
METABOLISM Glucose
LIVER FUNCTION :
A) HEPATOCELLULAR
B) HEPATOBILIARY Alkaline phosphatase (ALP)
SD17 F ↑ = 1.9 G3
Aspartate aminotransferase (AST or
SGOT)
Alanine aminotransferase (ALT or SGPT)
Tota l bilirubin
ACUTE PHASE REACTANTS Fibrinogen (also under
coagulation)**
KIDNEY FUNCTION
Crea tinine
Blood Urea Nitrogen (BUN)
OTHERS
(ACID/BASE BALANCE ,
CHOLINESTERASES ,
HORMONES , LIPIDS ,
METHEMOGLOBIN , AND
PROTEINS ) Albumin (A)*
GLOB*
A/G ra tio*
A1A GP*
A2M*
Total protein
Carbon dioxide
Globulin
Fa sting triglycerides
Total Cholesterol
Creatine kinase (CK)
Gamma -GT
Lactate dehydrogenase (LDH)
* See table below. ** See table on page 16
Table 44: Serum chemistry results for males and females
Clinical chemistry results showed an increase in ALP levels in group 3 females at study day 17.
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72 Dosing phase
In groups 2 and 3 males and females, higher mean alpha -1 acid glycoprotein (A1AGP) and
alpha -2-macroglobu lin (A2M) and lower Albumin:Globulin (AG) ratios (primarily due to lower
albumin with slight contribution from higher globulins) on study days 4 and 17 were reported.
Dose (µg RNA/Dose Da y)
Parame ter Ma les Fem a les
Test Article Vehicle BNT162b2(V9) BNT162b3c Vehicle BNT162b2(V9) BNT162b3c
0 30 30 0 30 30
ALB (g/dL)
4D 3.98 0.93x 0.92x 4.16 0.86x 0.90x
17D 3.50 - - 3.60 0.85x 0.86x
GLOB (g/dL)
4D 2.13 - - 2.10 - 1.05x
17D 1.89 1.10x 1.07x 1.84 1.04x -
AG
4D 1.88 0.90x 0.90x 1.98 0.86x 0.85x
17D 1.85 0.89x 0.89x 1.96 0.82x 0.85x
A1AGP
4D 174.358 9.42x 13.49x 239.774 7.95x 6.99x
17D 47.672 38.51x 42.40x 95.959 15.55x 17.21x
A2M
4D 113.4 20.44x 34.99x 212.1 3.32x 4.18x
17D 14.0 70.76x 128.16x 33.1 15.74x 17.89x
Control m ean values and the ratio of the test article -related findings rela tive to control m eans are listed.
- = Not test a rticle rela ted; A1AGP = a lpha -1 a cid glycoprotein; A2M = a lpha -2-macroglobulin;
AG = Albumin/globulin ratio; ALB = Albumin; D = Day; GLOB = Globulin; TP = Protein, total.
Table 45: Test article -related clinical chemistry parameter effects (mean control values and ratio
relative to control mean)
Recovery phase
At study 22 (recovery), all test article related changes were fully reversed, with the exception of
higher globulins in group 2 males and groups 2 and 3 females, and lower AG ratio in group 2
females.
Dose (µg RNA/Dose Day)
Parame ter Ma les Fem a les
Test Article Vehicle BNT162b2(V9) BNT162b3c Vehicle BNT162b2(V9) BNT162b3c
0 30 30 0 30 30
GLOB (g/dL)
R22 2.10 1.08x - 2.26 1.06x 1.07x
AG
R22 1.76 - - 1.90 0.91x -
Control m ean values and the ratio of the test article -related findings rela tive to control m eans are listed.
- = Not test a rticle related; AG = Albumin/globulin ra tio; GLOB = Globulin; R = Recovery day.
Table 46: Test article -related clinical chemistry parameter effects (mean control values and ratio
relative to control mean)
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73 Other statistically significant or apparent differences between test article and control group
clinical chemistry parameters were not test article related due small magnitude of the difference and general overlap in magnitude of individual values with controls.
Hematology
HEMATOLOGY
MEASUREMENT
RELATED TO END POINTS DIFFERENT THAN
THE CONCURRENT CONTROL
(LIST THE ENDPOINT, STUDY
DAY (SD), SEX, DOSE GROUP (G),
DIRECTION, FOLD CHANGE if great
or less than 1. 525, ie, ≥ 1.6 or ≤ 1.6 Not of NOTE
Red blood cells
HCT (%)*
Mea n Corp. Hb. (MCH)* Mea n Corp. Hb. Conc. (MCHC)*
Mea n Corp. Hb. Conc. (MCHC)*
RDW%*
Reticulocyte*
Hemoglobin Conc. (Hb)
Mean Corp. Volume (MCV)
Tota l Erythrocyte Count (RBC)
White blood cells
Lymphocyte count
SD17 F ↑ = 1.7 G2
SD17 F ↑ = 1.8 G3
WBC*
Neutrophil*
Monocyte*
Eosinophil*
Ba sophil*
LUC* Ma crophage
Leukocytes
Clotting potential
Fibrinogen*
Activa ted partial -thromboplastin time
clotting time
Prothrombin time
Platelet count
Others
Bone marrow cytology
* See table on page 16
Table 47: Hematology results for males and females
Terminal phase
Hematology results showed an increase in lymphocyte levels in groups 2 and 3 females at study
day 17.
Test article -related hematology and coagulation findings were similar in groups 2 and 3.
However, higher mean white blood cell (WBC) counts and fibrinog en concentrations, lower (day
4) and higher (day 17) reticulocyte counts, and lower red blood cell mass (red blood cell count,
hemoglobin and hematocrit) were reported in groups 2 and 3 when compared to group 1. Higher
WBC primarily involved higher neutrophils, monocytes and large unstained cells. Higher
25 With rounding up at the tenth decimal place. Therefore, 1.54 or less becomes 1.5 and is not reported
and 1.55 or greater becomes 1.6 and is reported.
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74 eosinophils and basophils were also reported. They were present on days 4 and 17, with higher
counts on day 17 than day 4. On study day 17, there were also test article -related higher
fibrinogen concentrations in both sexes. Hyper -segmented neutrophils were present on peripheral
blood smears of test article -treated animals.
In addition, there were test article -related transiently lower reticulocyte counts on study day 4,
and higher reticulocytes on study da y 17 (females only). These changes were with attendant
expected changes in RBC indices (higher mean cell hemoglobin concentration; males on day 4; lower mean cell hemoglobin [MCH] and higher red cell distribution width on day 17; both sexes). These were as sociated with lower RBC mass on days 4 and 17 (comparable on both days
or slightly lower on day 17). Test article -related clinical chemistry findings were similar in
groups 2 and 3. However, higher mean alpha -1 acid glycoprotein and alpha -2-macroglobulin a nd
lower AG ratios (primarily due to lower albumin with slight contribution from higher globulins) were reported in males and females of both groups on days 4 and 17.
Recovery phase
After a 3 -weeks recovery phase, all test article-related hematology and coagulation changes were
fully reversed, with the exception of higher red cell distribution width.
There were no test article-related findings reported in urinalysis parameters in the dosing or
recovery phase.
Dose (µg RNA/Dose Day)
Parame ter Ma les Fem a les
Test Article Vehicle BNT162b2(V9) BNT162b3c Vehicle BNT162b2(V9) BNT162b3c
0 30 30 0 30 30
HCT (%)
4D 48.04 0.90x 0.91x 44.91 0.93x 0.93x
17D 42.61 0.90x 0.92x 41.67 0.91x 0.89x
MCH (pg)
4D 18.51 - - 18.37 - -
17D 18.27 0.96x - 18.62 0.97x 0.96x
MCHC (g/dL)
4D 31.24 1.04x 1.03x 32.34 - -
17D 32.46 - - 33.18 - -
RDW (%)
4D 12.27 - - 11.11 - -
17D 11.63 1.21x 1.18x 11.33 1.18x 1.18x
RETIC
(10e3/uL)
4D 392.1 0.27x 0.27x 301.7 0.43x 0.44x
17D 178.8 - - 168.9 1.31x 1.20x
WBC
(10e3/uL)
4D 7.60 1.41x 1.28x 6.01 1.30x 1.43x
17D 3.84 2.30x 2.24x 2.16 2.64x 2.95x
NEUT
(10e3/uL)
4D 1.083 2.28x 2.00x 0.920 2.51x 3.13x
17D 0.674 6.60x 6.46x 0.409 6.04x 7.04x
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75 Dose (µg RNA/Dose Day)
Parame ter Ma les Fem a les
Test Article Vehicle BNT162b2(V9) BNT162b3c Vehicle BNT162b2(V9) BNT162b3c
0 30 30 0 30 30
MONO
(10e3/uL)
4D 0.109 1.83x 1.96x 0.093 1.89x 2.52x
17D 0.071 3.30x 3.58x 0.056 2.75x 3.14x
EO (10e3/uL)
4D 0.081 - - 0.057 - 2.16x
17D 0.056 2.52x 2.18x 0.029 3.17x 3.34x
BASO
(10e3/uL)
4D 0.016 1.88x 2.31x 0.009 1.89x 2.67x
17D 0.003 5.67x 6.33x 0.001 8.00x 10.00x
LUC
(10e3/uL)
4D 0.046 4.07x 3.98x 0.030 4.20x 4.43x
17D 0.026 8.04x 12.42x 0.010 13.20x 19.00x
FIB (mg/dL)
17D 253.1 2.36x 2.39x 217.2 2.49x 2.59x
Control m ean values and the ratio of the test article -related findings rela tive to control m eans are listed.
- = Not test a rticle rela ted; BASO = Basophil, a bsolute; D = Day; EO = Eosinophil, absolute;
FIB = Fibrinogen; HCT = Hematocrit; LUC = La rge unstained cells, a bsolute; MCH = Mean cell hemoglobin;
MCHC = Mean cell hemoglobin concentration; MONO = Monocyte, a bsolute; NEUT = Neutrophil, a bsolute;
RDW = Red cell distribution width; RETIC = Reticulocyte, absolute; WBC = White blood cells.
Table 48: Test article -related hematology and coagulation parameter effects at main sacrifice
(mean control values and ratio relative to control mean)
Dose (µg RNA/Dose Day)
Pa ra meter Ma les Fem a les
Test Article Vehicle BNT162b2(V9) BNT162b3c Vehicle BNT162b2(V9) BNT162b3c
0 30 30 0 30 30
RDW (%)
R22 11.93 1.13x 1.12x 10.80 1.21x 1.23x
Control m ean values and the ra tio of the test article -related findings relative to control means are listed.
R = Recovery day; RDW = Red cell distribution width.
Table 49: Test article -related hematology and coagulation parameter effects at recovery phase
(mean control values and ratio relative to control mean)
Bone Marrow Assessment
Bone marrow smears were prepared for all animals and were not examined.
Systemic toxicity:
No treatment -related, mortality, nor any toxicologically relevant changes in clinical signs, body
weight, food consumption, body temperature, ophthalmic changes, or urinalysis were reported. Organ Weight:
In groups 2 and 3 males and females, test article-related organ weight differences included
higher absolute and relative (to body and brain weight) spleen weights were reported.
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76 No test article -related organ weight changes were reported at the end of the recovery phase.
Table of organ weights results f or males
BWT
ABS N
10 Mean
296 06 Ratio
R REF SD
16 40 N
10 Mean
271 17 Ratio
0 92 SD
17 12
† N
10 Mean
262 59 Ratio
0 89 SD
18 67
†
Brain ABS 10 1 9061 R REF 0 0899 10 1 9159 1 01 0 1445 10 1 9082 1 00 0 0599
OW:BW 10 0 6449 R REF 0 0335 10 0 7087 1 10 0 0664 † 10 0 7294 1 13 0 0481 †
OW:BRN 10 1 0000 R REF 0 0000 10 1 0000 1 00 0 0000 10 1 0000 1 00 0 0000
Epididymis ABS 10 1 1647 R REF 0 1713 10 1 0626 0 91 0 1281 10 1 0508 0 90 0 0665
OW:BW 10 0 3936 R REF 0 0536 10 0 3922 1 00 0 0428 10 0 4026 1 02 0 0442
OW:BRN 10 0 6112 R REF 0 0867 10 0 5570 0 91 0 0756 10 0 5512 0 90 0 0400
Gland, Adrenal ABS 10 0 0697 R REF 0 0068 10 0 0727 1 04 0 0149 10 0 0706 1 01 0 0107
OW:BW 10 0 0236 R REF 0 0021 10 0 0267 1 13 0 0045 10 0 0270 1 14 0 0044
OW:BRN 10 0 0366 R REF 0 0040 10 0 0383 1 04 0 0091 10 0 0371 1 01 0 0061
Gland, Prostate ABS 10 0 7215 R REF 0 1036 10 0 7324 1 02 0 2129 10 0 6755 0 94 0 1088
OW:BW 10 0 2439 R REF 0 0328 10 0 2699 1 11 0 0726 10 0 2575 1 06 0 0401
OW:BRN 10 0 3781 R REF 0 0476 10 0 3808 1 01 0 0941 10 0 3539 0 94 0 0556
Heart ABS 10 0 9152 R REF 0 0698 10 0 9242 1 01 0 1151 10 0 8795 0 96 0 1051
OW:BW 10 0 3097 R REF 0 0260 10 0 3405 1 10 0 0329 * 10 0 3346 1 08 0 0278
OW:BRN 10 0 4807 R REF 0 0388 10 0 4852 1 01 0 0758 10 0 4614 0 96 0 0583
Kidney ABS 10 2 1659 R REF 0 1836 10 2 2197 1 02 0 2229 10 2 0252 0 94 0 1974
OW:BW 10 0 7312 R REF 0 0411 10 0 8179 1 12 0 0507 † 10 0 7710 1 05 0 0495
OW:BRN 10 1 1356 R REF 0 0682 10 1 1600 1 02 0 0939 10 1 0607 0 93 0 0914
Liver ABS 10 8 3218 R REF 0 5205 10 7 7880 0 94 0 4860 * 10 7 5872 0 91 0 5920 †
OW:BW 10 2 8131 R REF 0 1435 10 2 8771 1 02 0 1801 10 2 8905 1 03 0 1234
OW:BRN 10 4 3681 R REF 0 2325 10 4 0850 0 94 0 3960 10 3 9783 0 91 0 3168 *
Spleen ABS 10 0 5951 R REF 0 0613 10 0 7700 1 29 0 1038 † 10 0 7984 1 34 0 0899 †
OW:BW 10 0 2008 R REF 0 0147 10 0 2842 1 42 0 0352 † 10 0 3051 1 52 0 0373 †
OW:BRN 10 0 3120 R REF 0 0264 10 0 4019 1 29 0 0431 † 10 0 4191 1 34 0 0521 †
Testis
ABS N
10 Mean
3 2727 Ratio
R REF SD
0 3106 N
10 Mean
3 4683 Ratio
1 06 SD
0 3109 N
10 Mean
3 2716 Ratio
1 00 SD
0 2275
OW:BW 10 1 1090 R REF 0 1254 10 1 2803 1 15 0 1001 * 10 1 2538 1 13 0 1447 *
OW:BRN 10 1 7171 R REF 0 1440 10 1 8123 1 06 0 1262 10 1 7146 1 00 0 1080
Thymus ABS 10 0 5914 R REF 0 0676 10 0 4673 0 79 0 0934 † 10 0 4200 0 71 0 0907 †
OW:BW 10 0 1999 R REF 0 0222 10 0 1718 0 86 0 0293 * 10 0 1591 0 80 0 0275 †
OW:BRN 10 0 3098 R REF 0 0266 10 0 2448 0 79 0 0507 † 10 0 2199 0 71 0 0460 †
Table 50: Male’s organ weight: Absolute weights are expressed as mean (grams). Entries in table
are expressed as organ weight from animals taken at the end of the terminal phase.
Body weight was decreased 11% in group 3 males. Spleen weight was increased 29% and 34%
in groups 2 and 3 males, respectively. Thymus weight was decreased 21% and 29% in groups 2
and 3 males, respectively.
Group Number:
Dose: REF
0 µg/day 2
30 µg/day 3
30 µg /day
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77 Table of organ weights results for females
BWT
ABS N
10 Mean
198 73 Ratio
R REF SD
10 80 N
10 Mean
194 56 Ratio
0 98 SD
10 69 N
10 Mean
191 82 Ratio
0 97 SD
7 14
Brain ABS 10 1 8610 R REF 0 0694 10 1 7868 0 96 0 0595 10 1 8407 0 99 0 0783
OW:BW 10 0 9383 R REF 0 0507 10 0 9203 0 98 0 0467 10 0 9604 1 02 0 0451
OW:BRN 10 1 0000 R REF 0 0000 10 1 0000 1 00 0 0000 10 1 0000 1 00 0 0000
Gland, Adrenal ABS 10 0 0882 R REF 0 0162 10 0 0886 1 00 0 0156 9@ 0 0907 1 03 0 0192
OW:BW 10 0 0442 R REF 0 0068 10 0 0454 1 03 0 0065 9@ 0 0471 1 07 0 0090
OW:BRN 10 0 0474 R REF 0 0088 10 0 0496 1 05 0 0085 9@ 0 0490 1 03 0 0100
Heart ABS 10 0 7450 R REF 0 0803 10 0 7573 1 02 0 0866 10 0 7173 0 96 0 0860
OW:BW 10 0 3749 R REF 0 0343 10 0 3893 1 04 0 0417 10 0 3736 1 00 0 0387
OW:BRN 10 0 4004 R REF 0 0418 10 0 4248 1 06 0 0563 10 0 3903 0 97 0 0491
Kidney ABS 10 1 5273 R REF 0 0808 10 1 6343 1 07 0 0778 * 10 1 6164 1 06 0 1416
OW:BW 10 0 7696 R REF 0 0415 10 0 8412 1 09 0 0418 † 10 0 8417 1 09 0 0529 †
OW:BRN 10 0 8216 R REF 0 0519 10 0 9153 1 11 0 0477 † 10 0 8787 1 07 0 0758
Liver ABS 10 5 4571 R REF 0 3313 10 5 6490 1 04 0 5559 10 5 8104 1 06 0 4922
OW:BW 10 2 7466 R REF 0 0920 10 2 9002 1 06 0 1853 * 10 3 0247 1 10 0 1541 †
OW:BRN 10 2 9329 R REF 0 1468 10 3 1630 1 08 0 3132 10 3 1580 1 08 0 2526
Ovary ABS 10 0 1167 R REF 0 0158 10 0 1053 0 90 0 0180 9@ 0 1113 0 95 0 0170
OW:BW 10 0 0588 R REF 0 0076 10 0 0542 0 92 0 0097 9@ 0 0579 0 98 0 0073
OW:BRN 10 0 0627 R REF 0 0079 10 0 0590 0 94 0 0101 9@ 0 0601 0 96 0 0085
Spleen ABS 10 0 4382 R REF 0 0669 10 0 6796 1 55 0 1031 † 10 0 6199 1 41 0 0555 †
OW:BW 10 0 2202 R REF 0 0294 10 0 3492 1 59 0 0489 † 10 0 3231 1 47 0 0261 †
OW:BRN 10 0 2353 R REF 0 0333 10 0 3803 1 62 0 0550 † 10 0 3374 1 43 0 0337 †
Thymus ABS 10 0 4588 R REF 0 0700 10 0 3967 0 86 0 1131 10 0 3906 0 85 0 0582
OW:BW 10 0 2310 R REF 0 0336 10 0 2031 0 88 0 0583 10 0 2036 0 88 0 0288
OW:BRN 10 0 2469 R REF 0 0386 10 0 2221 0 90 0 0655 10 0 2127 0 86 0 0324
Table 51: Female’s organ weight: Absolute weights are expressed as mean (grams). Entries in
table are expressed as organ weight from animals taken at the end of the terminal phase.
Spleen weight was increased 55% and 41% in groups 2 and 3 females, respectively. Thymus
weight was decreased 14% and 15% in groups 2 and 3 females, respectively.
Gross pathology:
Dosing phase
In groups 2 and 3, large draining lymph nodes (abnormal size, enlarged) and dark/pale and/or firm injection sites (abnormal color, dark/pale and/or abnormal consistency, firm) were reported. In group 3 females, large spleen and inguinal lymph nodes (abnormal size, enlarged) were reported. Group Number:
Dose: REF
0 µg/day 2
30 µg/day 3
30 µg /day
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78
Recovery pha se
In one group 2 males and one group 3 females, large draining lymph nodes (abnormal size,
enlarged) were reported. Large inguinal lymph nodes (abnormal size, enlarged) were reported in
one group 3 females, indicating a partial recovery of these findings. In groups 2 and 3 males and females, pale/dark and/or firm injection sites and enlarged spleen were not reported at the end of recovery phase, indicating a complete recovery of these findings.
Microscopic findings:
Terminal sacrifice
In groups 2 and 3 males and females, findings at the injection site (mixed cell inflammation and
edema), draining and inguinal lymph nodes (increased cellularity, plasma cells and germinal centers), liver (hepatocellular vacuolation), spleen (increased cellularity, hemat opoietic cells and
germinal centers), and bone marrow (increased cellularity, hematopoietic cells) were reported.
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Group Number:
Dose:
No. Animals Per Dose Group: Male 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10 Female 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10
EYE Number Examined 10 10 10 10 10 10
Unremarkable 10 10 10 9 9 8
Mineralization, Cornea - - - - 1 -
Minimal - - - - 1 -
Rosettes retina - - - 1 - 2
Minimal - - - 1 - 2
GLAND, ADRENAL Number Examined 10 10 10 10 10 10
Unremarkable 10 10 10 10 10 9
Hypertrophy, Cortex - - - - - 1
Present - - - - - 1
GLAND, HARDERIAN Number Examined 10 10 10 10 10 10
Unremarkable 10 10 10 6 9 7
Degeneration/Necrosis - - - 2 - 2
Minimal - - - 2 - 2
Infiltration mononuclear cell
- - - 3 1 1
Minimal - - - 3 1 1
GLAND, PITUITARY Number Examine d
Unrema rk ab le
Cyst
Min im a l 10 10 10 10 10 10
10 9 8 9 10 8
- 1 2 1 - 2
- 1 2 1 - 2
GLAND, PROSTATE Number Examined
Unrema rk ab le
Infiltration mononuclear cell
Min im a l 10 10 10 - - -
10 10 9 - - -
- - 1 - - -
- - 1 - - -
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80 Group Number:
Dose:
No. Animals Per Dose Group: Male 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10 Female 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10
GLAND, SALIVARY Number Examined
Unrema rk ab le
Hypertrophy
Min im a l 10 10 10 10 10 10
10 10 10 9 10 10
- - - 1 - -
- - - 1 - -
GUT -ASSOCIATED LYMPHOID TISSUE Number Examined 10 10 10 8 10 1
Unremarkable 10 10 10 8 9 10
Mineralization, Germinal center - - - - 1 -
Minimal - - - - 1 -
HEART Number Examined 10 10 10 10 10 10
Unremarkable 10 10 10 10 10 10
JOINT Number Examined 10 10 10 10 10 10
Unremarkable 10 7 10 9 8 7
Inflammation, Extra -capsular - 3 - - 2 3
Minimal - 3 - - 2 3
Physeal dysplasia - - - 1 - -
Minimal - - - 1 - -
KIDNEY Number Examined 10 10 10 10 10 10
Unremarkable 9 9 9 8 6 10
Tubular basophilia - 1 - - 1 -
Minimal - 1 - - 1 -
Infiltration mononuclear cell - - 1 2 3 -
Minimal - - 1 2 3 -
Dilatation, Pelvis 1 - - - - -
Minimal 1 - - - - -
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81 Group Number:
Dose:
No. Animals Per Dose Group: Male 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10 Female 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10
LARGE INTESTINE, COLON Number Examined
Unrema rk ab le
Infiltration mixed cell, Mucosa
Min im a l 10 10 10 10 10 10
10 10 10 10 10 9
- - - - - 1
- - - - - 1
LIVER Number Examined
Unremarkable
Vacuolation, Hepatocyte; Periportal
Min im a l 10 10 10 10 10 10
10 5 3 10 0 3
- 5 7 - 10 7
- 5 7 - 10 7
LUNG Number Examined
Unremarkable
Infiltration mixed cell
Min im a l 10 10 10 10 10 10
10 10 10 9 9 10
- - - 1 1 -
- - - 1 1 -
LYMPH NODE, DRAINING Number Examined
Unrema rk ab le
Increased cellularity, Plasma cell
Min im a l
Mild
Moderate
Increased cellularity, Germinal center
Min im a l
Mild 10 9 10 10 10 10
8 1 1 8 1 1
- 7 8 - 9 7
- 1 4 - 1 1
- 4 3 - 1 5
- 2 1 - 7 1
2 6 8 2 5 6
1 2 2 1 3 4
1 4 6 1 2 2
LYMPH NODE, INGUINAL Number Examined
Unrema rk ab le
Increased cellularity, Germinal center
Min im a l
Mild
Increased cellularity, Plasma cell
Min im a l 9 10 10 10 10 10
8 5 4 9 4 1
1 5 6 1 6 9
- 1 1 1 3 6
1 4 5 - 3 3
- 1 1 - 2 4
- 1 1 - 2 4
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82 Group Number:
Dose:
No. Animals Per Dose Group: Male 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10 Female 1 2
0 µg/day 30 µg/day
10 10 3
30 µg
/day
10
PANCREAS Number Examined 10 10 10 10 10 10
Unremarkable 10 10 10 10 6 10
Atrophy, Acinar cell - - - - 4 -
Minimal - - - - 4 -
Infiltration mononuclear cell, Interstitium - - - - 1 -
Minimal - - - - 1 -
SITE, INJECTION Number Examined 10 10 10 10 10 10
Unremarkable 6 0 0 5 0 0
Inflammation 4 10 10 5 10 10
Min im a l 4 - - 5 - -
Mild - 7 5 - 7 9
Moderate - 3 5 - 3 1
Edema - 9 9 - 10 10
Mild - 8 8 - 9 9
Moderate - 1 1 - 1 1
SPLEEN Number Examined
Unremarkable
Increased cellularity, Germinal center
Min im a l
Increased cellularity, Hematopoietic cell
Min im a l 10 10 10 10 10 10
10 0 0 10 0 0
- 5 5 - 6 5
- 5 5 - 6 5
- 10 10 - 9 10
- 10 10 - 9 10
STOMACH Number Examined
Unremarkable
Infiltration mononuclear cell, Serosa
Min im a l
Erosion
Min im a l 10 10 10 10 10 10
10 10 9 10 9 10
- - - - 1 -
- - - - 1 -
- - 1 - - -
- - 1 - - -
Table 52: Microscopic findings at terminal sacrifice
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Recovery sacrifice
A complete recovery of most of the findings reported at the terminal phase. Inflammation at the
injection site was characterized by mostly lymphocytes and plasma cells with few neutrophils
(indicating partial recovery) and no edema (full recovery). In groups 2 and 3 males and females,
increased cellularity of the germinal centers in the spleen partially recovered, as the incidence and/or severity of these findings were lower in recovery phase animals as compared with dosing phase animals. At the end of recovery phase, mature plasma cells had replaced the plasma blasts
identified in the inguinal and draining lymph nodes in the dosing phase animals. Infiltration of
macrophages was reported in the draining lymph nodes (minimal to mild) in groups 2 and 3 males and females and in the inguinal lymph nodes (minimal) of group 2 males and females.
Dermal Assessment
Dosing phase
In all group 2 (except animal #17) animals, related injection site edema grade 2 (slight, edges of
area well defined by definite raising) or grade 3 (moderate, raised approximately 1 mm) were
reported following dosing on days 1, 8 and/or 15. The edema was generally reported up to 72 hours post dose, and fully resolved prior to dose administration on days 8 and 15. In all group 2 (except animals 16 -21 and 30) animals, erythema was also reported at the injection site,
following each dose administration. However, it was only a grade 1 (very slight, barely perceptible) and fully resolved prior to the next dose administration.
In all gro up 3 animals, injection site edema grade 2 (slight, edges of area well defined by
definite raising) or grade 3 (moderate, raised approximately 1 mm) were reported following dosing on days 1, 8 and/or 15. The edema was generally reported up to 72 hours post dose, and
fully resolved prior to dose administration on days 8 and 15. In all group 3 (except animal 39) animals, erythema was also reported at the injection site, following each dose administration.
However, it was only a grade 1 (very slight, barely perceptible) and fully resolved prior to the
next dose administration.
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84 Group mean dermal assessment data are listed in the table below:
Male
Parameter
Phase
Day
Group
N
Mean Sta ndard
Devia tion Pa irwise
p-value
Edema - Left Dosing 1 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.63 0.51 0.001 **
3: BNT162b3c 15 0.80 0.55 0.001 **
Dosing 8 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 1.19 0.51 0.001 **
3: BNT162b3c 15 1.43 0.12 0.001 **
Dosing 15 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 1.33 0.45 0.001 **
3: BNT162b3c 15 1.54 0.46 0.001 **
Male
Parameter
Phase
Day
Group
N
Mean Sta ndard
Devia tion Pa irwise
p-value
Erythema - Left Dosing 1 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.03 0.13 0.682
3: BNT162b3c 15 0.04 0.13 0.270
Dosing 8 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.23 0.27 0.001 **
3: BNT162b3c 15 0.41 0.17 0.001 **
Dosing 15 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.00 0.00 0.999
3: BNT162b3c 15 0.09 0.20 0.050 *
Table 53: Edema and erythema findings in males at study days 1, 8, and 15
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85 Female
Parameter
Phase
Day
Group
N
Mean Sta ndard
Devia tion Pa irwise
p-value
Edema - Left Dosing 1 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 1.28 0.57 0.001 **
3: BNT162b3c 15 1.08 0.58 0.001 **
Dosing 8 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 1.44 0.23 0.001 **
3: BNT162b3c 15 1.47 0.28 0.001 **
Dosing 15 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 1.64 0.34 0.001 **
3: BNT162b3c 15 1.78 0.27 0.001 **
Female
Parameter
Phase
Day
Group
N
Mean Sta ndard
Devia tion Pa irwise
p-value
Erythema - Left Dosing 1 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.56 0.38 0.001 **
3: BNT162b3c 15 0.66 0.17 0.001 **
Dosing 8 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.50 0.09 0.001 **
3: BNT162b3c 15 0.58 0.11 0.001 **
Dosing 15 1: Sa line 15 0.00 0.00 REF
2: BNT162b2 (V9) 15 0.33 0.22 0.001 **
3: BNT162b3c 15 0.60 0.14 0.001 **
Table 54: Edema and erythema findings in females at study days 1, 8, and 15
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86 Male
Parameter
Phase
Group
N
Mean Sta ndard
Devia tion
Edema - Left Recovery 2: BNT162b2 (V9) 4 1.08 0.17
3: BNT162b3c 5 0.80 0.18
Erythema - Left Recovery 2: BNT162b2 (V9) 4 0.00 0.00
3: BNT162b3c 5 0.00 0.00
Female
Parameter
Phase
Group
N
Mean Sta ndard
Devia tion
Edema - Left Recovery 2: BNT162b2 (V9) 5 1.07 0.15
3: BNT162b3c 5 1.13 0.18
Erythema - Left Recovery 2: BNT162b2 (V9) 5 0.13 0.18
3: BNT162b3c 5 0.33 0.24
Table 55: Edema and erythema findings in males and females at recovery phase
Body temperature:
No test article -related effects on body temperature was reported.
Urinalysis:
There were no test article-related findings on urinalysis. Due to small magnitude of the
difference and general overlap in magnitude of indivi dual values with controls, all statistically
significant or apparent differences in urinalysis parameters between groups 2 and 3 and control
group were not test article related.
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87
Serology:
Microneutralization (MN) assay for serological detection of SARS -CoV-2 specific neutralizing
antibodies in animal sera were used. This is relative to the "work order 4" agreed between
VisMederi Sri and Pfizer. The MN -CPE (Microneutralization based on Cytopathic effect)
method is a specific technique used for the identification of virus -specific neutralizing antibodies
against live viruses which are able to prevent the virus infection. The following table shows
geometric mean titers for grouped subjects by s ex and for vaccine administered.
Study Day Sex saline 30µg
BNT162b2(V9) 30µg
BNT162b3c
PIO Day 8
(Day -5) Male 5 5 5
Female 5 5 5
Day 17 Male 5 1114 993
Female 5 2501 1810
R:P Day 21
(Day 38) Male 5 5120 3880
Female 5 5120 3880
PIO = prior to dose initiation; RP = Recovery phase
Table 56: Geometric mean titers (GMTs) for each dose group by sampling day and sex
In groups 2 and 3, SARS -CoV-2 neutralizing antibody responses in males and females at the end
of the dosing (day 17) and recovery phases (day 21) were reported. SARS -CoV-2 neutralizing
antibody responses were not reported in animals prior to vaccine administration or in group 1 (control) animals.
Test article related effects are listed in the table below :
Test article related effects
↓ Albumin
↑ Globulin ↓ AG ratio
↓ Reticulocytes
↑ Monocytes
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88 Test article related effects
↑ Neutrophils
↑ Eosinophils
↑ Basophils
↑ WBC
↑ LUC
↑ Fibrinogens
↑ Red cell distribution width (RDW%)
↑ Alpha1-acid glycoproteins
↑ Alpha2-macroglobulins
↑ Spleen weight
↓ Thymus weight for females
Injection site findings (mixed cell inflammation and edema)
Draining and inguinal lymph nodes findings (increased
cellularity, plasma cells and germinal centers)
Liver findings (hepatoce llular vacuolation)
Spleen findings (increased cellularity, hematopoietic cells
and germinal centers)
Bone marrow (increased cellularity, hematopoietic cells)
Immune responses in groups 2 and 3
Assessment:
No treatment -related, mortality, nor any toxicologically relevant changes in clinical signs, body
weight, food consumption, body temperature, ophthalmic changes, or urinalysis were reported. Minimal decreases in globulin concentration was reported in both sexes from groups 2 and 3. Concurrently, minimally increased albumin was reported. Hence, the albumin to globulin ratio
was lower in both males and females from groups 2 and 3. These changes indicate an acute phase
response/inflammation. These changes were not reported in the recovery animals. Reticulocytes are immature red blood cells (RBCs). In the process of erythropoiesis (red blood
cell formation), reticulocytes develop and mature in the bone marrow and the n circulate for
about a day in the blood stream before developing into mature red blood cells. Like mature red blood cells, in mammals, reticulocytes do not have a cell nucleus .
26 Abnormally low numbers of
reticulocytes can be attributed to chemotherapy , aplastic anemia , pernicious anemia , bone
marrow malignancies, problems of erythropoietin production, various vitamin or mineral
deficiencies ( iron, vitamin B 12, folic acid ), disease states ( anemia of chronic disease ) and other
causes of anemia due to poor RBC production.27
Monocytosis could be indicative of the intended immune response or could be secondary to
muscle damage at the site of injection as an indication of inflammation and repair. The increases in the monocyte count might be related to test article treatment.
26 https://en.wikipedia.org/wiki/Reticulocyte
27 https://www.uofmhealth.org/health -library/hw203366
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89 Neutrophils are key components in the system of defense against infection. An individual with
absence or scarcity of neutrophils (neutropenia) is vulnerable to infection. The increase in neutrophils might be related to the immune responses initiated by the test article treatment.
Eosinophils are one of the immune system components responsible for combating multicellular parasites and certain infections in vertebrates. They are granulo cytes that develop during
hematopoiesis in the bone marrow before migrating into blood.
Basophils play a role in both parasitic infections and allergies. Basopenia has been reported in association with autoimmune urticaria.
White blood cells (WBCs ) (also called leukocytes or leucocytes) are the cells of the immune
system that are involved in protecting the body against both infectious disease and foreign
invaders. All white blood cells are produced and derived from multipotent cells in the bone
marrow known as hematopoietic stem cells . Leukocytes are found throughout the body,
including the blood and lymphatic sys tem.
28 The increase in WBC might be related to the
immune response induced by the test article treatment.
LUC is a measurement of the large, peroxidase -negative cells which cannot be further
characterized (i.e. as large lymphocytes, virocytes, or stem cells) present in a biological specimen. In LUC are found large lymphoid cells , more immature lymphocytes and other ce lls.
If the value is higher than normal, blood counts should be checked under a microscope slide.
The increases in fibrinogen levels were not considered frank toxicity but rather an anticipated
effect associated with an immunological response.
A red cell distribution width (RDW) test is a measurement of the range in the volume and size of red blood cells (erythrocytes). Red blood cells move oxygen from lungs to every cell in the body. The RDW blood test is often part of a complete blood count (CBC), a test that measures many
different components of the blood, including red cells. The RDW test is commonly used to
diagnose anemia, a condition in which the red blood cells can't carry enough oxygen to the rest
of the body. The RDW test may also be used to diagnose
29:
1- Other blood disorders such as thalassemia , an inherited disease that can cause severe
anemia
2- Medical conditions such as heart disease, diabetes , liver disease , and cancer , especially
colorectal cancer .
Alpha -1-acid glycoprotein (α 1AGp,30 AGP or AAG ), which is modulated by two polymorphic
genes , is an acute phase ( acute phase protein ) plasma alpha -globulin glycoprotein . It has a
normal plasma concentration between 0.6 -1.2 mg/mL (1 -3% plasma protein) and is synthesized
28 Maton, D., Hopkins, J., McLaughlin, Ch. W., Johnson, S., Warner, M. Q., LaHart, D., & Wright, J. D., Deep V.
Kulkarni (1997) . Human Biology and Health. Englewood Cliffs, New Jersey, US: Prentice Hall. ISBN 0-13-9 81176 -
1.
29 https://medlineplus.gov/lab -tests/rdw -red-cell-distribution -width/
30 https://en.wikipedia.org/wiki/Orosomucoid#cite_note -loganabbrev -1
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90 primarily in hepatocytes (5). Plasma levels are affected by pregnancy, burns, certain drugs, and
certain diseases, particularly HIV (5). The function of alpha -1-acid glycoprotein is to act as a
carrier of basic and neutrally charged lipophilic compounds. It is known as the primary carrier of
basic (positively charged) drugs (whereas albumin carries acidic (negatively charged) and neutral
drugs), steroids , and protease inhibitors (5, 6). AGP shows a complex interaction with thyroid
homeostasis. Alpha -1-acid glycoprotein (in low concentrations) was reported to stimulate the
thyrotropin (TSH) receptor and intracellular accumulation of cyclic AMP . However, high AGP
concentrations inhibited TSH signaling (7, 8). Alpha -1-acid glycoprotein has been identified as
one of four potentially useful circulating biomarkers for estimating the five -year risk of all-cause
mortality (the other three are albumin , very low -density lipoprotein particle size, and citrate ) (9).
Alpha -1-acid glycoprotein increases in obstr uctive jaundices while diminishes in hepatocellular
jaundice and in intestinal infections .31
Alpha -2-macroglobulin (α2M) is a large plasma protein found in the blood, mainly produced by
the liver, and also locally synthesized by macrophages , fibroblasts , and adrenocortical cells . It
acts as an antiprotease and is able to inactivate an enormous variety of proteinases. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein and as an inhibitor of
coagulat ion by inhibiting thrombin. Because it also binds to numerous growth factors and
cytokines, such as platelet -derived growth factor, basic fibroblast growth factor, TGF -β, insulin,
and IL -1β, it may act as a carrier protein. In the nephrotic syndrome when other lower molecular
weight proteins are lost in the urine, the concentration of alpha -2-macroglobulin rises 10 -fold or
more
32.
In groups 2 and 3, all clinical pathology findings (type and magnitude) were generally similar,
and consistent with expected immune responses to vaccines or secondary to inflammation. In both sexes, the main findings were present on days 4 and/or 17 and included higher acute phase proteins (alpha -1 acid glycoprotein; 7.0x -42x controls], alpha -2-macroglobulin (3.3x -128x] and
fibrinogen [2.4x -2.6x]) and white blood cell count (1.28x -2.95x; primarily involving neutrophils,
monocytes and large unstained cells, which typically represent large mononuclear cells) and lower albumin:globulin (0.90x-0.82x). On peripheral blood smears, hyper -segmented neutrophils
present and were considered to be secondary to the robust increases in neutrophil counts and
likely related to mobilization of bone marrow storage neutrophils and prolonged neutrophil
lifespan in circulation (10). These findings were consistent with the immune responses to vaccines.
Spleen weight increase might be related to the intended immune response. The spleen plays
important roles in regard to red blood cells and the immune system
33. It removes old red blood
cells and holds a reserve of blood in case of hemorrhagic shock while also recycling iron. As a
part of the mononuclear phagocyte system, it metabolizes hemoglobin removed from senescent
erythrocytes. The globin portion of hemoglobin is degraded to its constitutive amino acids, and
the heme portion is metabolized to bilirubin, which is subsequently shuttled to the liver for
31 https://en.wikipedia.org/wiki/Orosomucoid
32 https://en.wikipedia.org/wiki/Alpha -2-Macroglobulin
33 Spleen, Internet Encyclopedia of Science.
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91 removal34. It synthesizes antibodies in its white pulp and removes antibody-coated bacteria along
with antibody-coated blood cells by way of blood and lymph node circulation.
The thymus is a specialized primary lymphoid organ of the immune system . Within the thymus,
T cells or T lymphocytes mature. T cells are critical to the adaptive immune system , where the
body adapts specifically to foreign invaders. The thymus is composed of two identical lobes and
is located anatomically in the anterior superior mediastinum , in front of the heart and behind the
sternum .35 One of the major characteristics of vertebrate immunology is thymic involution , the
shrinking of the thymus with age, resulting in changes in the architecture of the thymus and a
decrease in tissue mass.36 T-cells are named for the thymus where T-lymphocytes migrate from
the bone marrow to mature. Its regression has been linked to the reduction in
immunosurveillance in the elderly.37
Test article -related injection site findings (mixed cell inflammation and edema) were reported.
Inflammation is a relatively common occurrence as part of the acute phase response following
administration of some vaccines.
The microscopic findings include d minimally increased cellularity of hematopoietic cells
(primarily myeloid) in the bone marrow and the spleen, minimal to moderate mixed cell
inflammation at the injection site and increased cellularity in germinal centers of lymphoid
organs. In addition, lower reticulocyte counts on day 4 (0.44x-0.27x), and higher reticulocytes on
day 17 (1.20x-1.31x; females on
…[truncated]