Document text
BNT162b2
Module 2.6.6. Toxicology Written Summary
CONFIDENTIAL
Page 1MODULE 2.6.6 TOXICOLOGY WRITTEN S UMMARY
This document contains confidential information belonging to BioNTech /Pfizer. Except as may be
otherwise agreed to in writing, by accepting or reviewing these materials, you agree to hold such
information in confidence and not to disclose it to others (except w here required by applicable
law), nor to use it for unauthorized purposes. In the event of actual or suspected breach of this
obligation, BioNTech /Pfizer should be promptly notified.
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Module 2.6.6. Toxicology Written Summary
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Page 2TABLE OF CONTENTS
LIST OF ABBREVIATION S AND DEFINITION OF TERMS ................................ .............. 3
2.6.6. TOXICOLOGY WR ITTEN SUMMARY................................ ................................ .......5
2.6.6.1. Brief Summary ................................ ................................ ................................ ....5
Table 2.6.6 -1. Overview of Toxicity Testing Program ................................ ................... 6
2.6.6.1.1. Test Article ................................ ................................ ............................. 7
2.6.6.1.2. Animals................................ ................................ ................................ ...8
2.6.6.2. Single -Dose Toxicity ................................ ................................ ........................... 8
2.6.6.3. Repeat -Dose Toxicity ................................ ................................ .......................... 8
2.6.6.3.1. Repeat -Dose Toxicity Study of Three LNP -Formulated RNA
Platforms Encoding for Viral Proteins by Repeated Intramuscular
Administration to Wistar Han Rats ................................ ................................ ....8
2.6.6.3.2. 17 -Day Intramuscular Toxicity Study of BNT162b2 (V9) in
Wistar Han Rats With a 3 -Week Recovery ................................ ..................... 11
2.6.6.4. Genotoxicity ................................ ................................ ................................ ......14
2.6.6.5. Carcinogenicit y ................................ ................................ ................................ .14
2.6.6.6. Reproductive and Developmental Toxicity................................ ....................... 14
2.6.6.6.1. A Combined Fertility and Developmental Study (Including
Teratogenicity and Postnatal Investigations) of BNT162b1, BNT162b2
and BNT162b3 b y Intramuscular Administration in the Wistar Han Rat ....... 15
2.6.6.7. L ocal Tolerance ................................ ................................ ................................ .16
2.6.6.8. Other Toxicity Studies (if available) ................................ ................................ .16
2.6.6.8.1. Antigenicit y ................................ ................................ .......................... 16
2.6.6.8.2. I mmunotoxicity ................................ ................................ ..................... 16
2.6.6.9. Discussion and Conclusions................................ ................................ .............. 16
2.6.6.10. References ................................ ................................ ................................ .......17
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Module 2.6.6. Toxicology Written Summary
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Page 3LIST OF ABBREVIATION S AND DEFINITION OF TERMS
A:G Albumin:globulin ratios
ALP Alkaline phosphatase
ALT Alanine aminotransferase
AST Aspartate aminotransferase
BASO Basophils
CBER Center for Biologics Evaluation and Research
CoV Coronavirus
COVID -19 Coronavirus Disease 2019
DART Developmental and Reproductive Toxicology
DSPC 1,2-distearo yl-sn-glycero-3-phosphocholine
ELISA Enzy me-linked immunosorbent assay
EOS Eosinophils
F0 Parental generation administered vaccine
F1 First generation offspring of F0 generation
GD Gestation day
GGT Gamma -glutam yl transferase
GLP Good Laboratory Practice
HCT Hematocrit
HGB Hemoglobin
IFN Interferon
IgG Immunoglobulin G
IL Interleukin
IM Intramuscular(ly )
LNP Lipid -nanop article
LUC Large unstained cells
modRNA Nucleoside -modified mRNA
MONO Monocy tes
mRNA Messenger RNA
NEUT Neutrophils
NHP Nonhuman primate
OECD Organisation for Economic Co -operation and Development
P2 S Spike protein P2 mutant
PLT Platelet
PND Postnatal day
RBC Red blood cells
RBD Receptor binding domain
RETI C Reticulocy tes
RNA Ribonucleic acid
S SARS -CoV -2 spike gl ycoprotein
SARS Severe Acute Respiratory S yndrome
SARS -CoV -2 S evere acute respiratory syndrome coronavirus 2; coronav irus causing
COVID -19
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Module 2.6.6. Toxicology Written Summary
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Page 4LIST OF ABBREVIATION S AND DEFINITION OF TERMS
TBIL Bilirubin, total
TNF Tumor necrosis factor
V8 Variant 8; P2 S
V9 Variant 9; P2 S
WBC White blood cells
WHO World Health Organization
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Page 52.6.6. TOXICOLOGY WRITTEN S UMMARY
2.6.6.1. Brief Summary
Pfizer and BioNTech havedeveloped a vaccine intended to prevent COVID- 19 that is caused
by SARS -CoV -2.The vaccine isbased on RNA encoding theSARS -CoV -2 S glycoprotein
antigen ,which is formulated in L NP, and is referred to as BNT162b2 vaccine candidate
(BioNTech code number BNT162, Pfizer code number PF- 07302048).
The nonclinical toxicity assessment of the BNT162b2 vaccine candidate consists of 3GLP-
compliant studies in Wistar Han rats including 2 pivotal repeat -dose toxicity studies and a
combined fertility and developmental study (Table 2.6.6 -1and Tabulated Summary 2.6.7.1 ).
Multiple vaccine candidates were evaluated in the nonclinical safet y studies; however, the
focus will be on the result sforBNT162b2 (V9) , the vaccine advance dinto the Phase 2/3
clinical trial and the subject of this application, and its variant BNT162b2 (V8) , which was
not administered clinically .BNT162b2 (V9) differs from BNT162b2 (V8) only in the
optimized codons used to improve antigen expression , but the amino acid sequences of the
encoded antigen are the same.
The design of the nonclinical repeat -dose toxicity stud ieswasconsistent with the WHO
Guidelines on Nonclinical Evaluation of Vaccines, the EMA Note for Guidance on
Preclinical Pharmacological and Toxicological Testing of Vaccines, and Japan guidance on
the nonclinical safety assessment of vaccines. In addition, the 2020 CBER guidance on
“Development and L icensure of Vaccines to Prevent COVI D-19 ” (US HHS, 2020 ) was
considered when assembling the nonclinical safet y licensure package as well as feedback
from regulatory agencies. All GL P-compliant studies were conducted in accordance with
Good Laboratory Practice fo r Nonclinical Laboratory Studies, Code of US Federal
Regulations (21 CFR Part 58), in an OECD Mutual Acceptance of Data member state. All
nonclinical studies described herein were conducted by or for Pfizer Inc or BioNTech RNA
Pharmaceuticals GmbH . The loc ation of records for inspection is included in each final study
report.
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Page 6Table 2.6.6 -1. Overview of Toxicity Testing Program
StudyaStudy
(Sponsor)
No.Dose Group
(µg RNA)Total
Volume
(µL)bNo. of
Animals/
GroupTabulated
Summary
Repeat -Dose Toxicity
17-Day, 2 or 3 Dose
(1Dose/Week) IM Toxicity
With a 3 -Week Recovery
Phase in Rats38166 Controlc
(0)
BNT162a1
(30)
BNT162a1
(30)
BNT162b1
(30)
BNT162b1
(100)
BNT162c1
(30)
BNT162b2 (V8)d
(100)200e
60
20
60
200e
70
200e15/sex
15/sex
15/sex
15/sex
15/sex
15/sex
15/sex2.6.7.7A
17-Day, 3 Dose
(1 Dose/Week)
IM Toxicity With a
3 Week Recovery Phase in
Rats20GR142 Salinef
(0)
BNT162b2 (V9)d
(30)
BNT162b3g
(30)60
60
6015/sex
15/sex
15/sex2.6.7.7B
Reproductive & Developmental Toxicity
IM Combined Fertility and
Developmental (Including
Teratogenicity and Postnatal
Investigations) Toxicity in
Rats20256434
(RN9391
R58)Salinef
(0)
BNT162b1
(30)
BNT162b2 (V9)d
(30)
BNT162b3
(30)60
60
60
6044F
44F
44F
44F2.6.7.12
a. All studies are GLP -compliant and were conducted in an OECD mutual acceptance of data -compliant
member state.
b. Doses w ere administered as 1 application at 1 site unless otherwise indicated.
c. Phosphate buffered saline, 300 mM sucrose.
d. Bold text highlighting the BNT162b2 vaccine candidate s.
e. One application (100 µL) at 2 sites for a total dose volume of 200 µL.
f. Sterile saline (0.9% NaCl).
g. BNT162b3 is also referred to as BNT162b3c in study reports.
In therepeat -dose toxicity stud ies, 30 or 100 µg BNT162b2 was tolerated when administered
once weekl y for a total of 3 IM doses. T here were no vaccine -related clinical signs or
mortalities observed. The vaccine induced a ninflammatory response which manifested as
increases in ty pical inflammatory blood parameters such as fibrinogen , acute phase protein s,
white blood cells (including NEUT, EOS, BASO, MONO, and/orLUC ), local injection site
reactions, transient increases in body temperature compared with controls, and microscopic
inflammation at the injection site, which sometimes extended into the surrounding tissues .
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Page 7Effects considered secondary to immune activation and the inflammatory response included a
reversible reduction in body weight post immunization without affecting body weight gain
between immunization s, transient decreases in RETI C, minimal decrease in RBC mass
parameters, and slight decreases in PL T. Evidence of an immune response was observed not
only in antigen -binding IgG and serum neutralizing response, but also as enlargement and
increase d cellularit y of germinal centers in the draining (iliac) lymph node. Responses to
inflammation were manifested as increased cellularity in the bone marrow and increased
extramedullary hematopoiesis in the spleen , whi ch w ereassociated with macroscopic
increased spleen size and increased absolute and relative spleen weight .
There were twovaccine -related nonadverse observations relevant to the liver. First, plasma
activity of GGT was elevated in comparison to the contr ol group. There was no elevation in
ALP or TBIL and no macroscopic or microscopic findings consistent with cholestasis or
hepatobiliary injury . Of note, elevation ofGGT was not replicated with BNT162b2 ( V9)in
the second repeat -dose toxicity study (Study 20GR142 ).Second, a nonadverse, reversible
vacuolation of portal hepatocy tes was present in animals administered BNT162b2, which
was not associated with alterations in hepatic function (eg, no elevations in AL Tor AST ).
This change may be related to hepatic distribution of the lipidsin the L NP
(Sedic etal,2018 ).
No new findings were observed during the recovery phase. At the end of the recovery , all
vaccine induced effects on local tolerance andbody weight were fully reverse dand most
clinical pathology parameter changes had resolved . Macroscopic and microscopic findings
had partial or complete recovery , although some animals treated with BNT162b2 s till had
enlarged iliac l ymph nodes and minimal to mild inflammation observed m icroscopicall yat
the injection site at the end of the recovery phase .
In the combined fertility and developmental study, administration of 4 I M doses (twice
before mating and t wice during gestation) of BNT162b2 at 30 µ g RNA/dosing day was
associated with nonadverse effects (body weight, food consumption and effects localized to
the injection site) after each dose administration in F0 female rats . There were no
BNT162b2 -related effects on mating performance or fertility in F0 female rats or on embry o-
fetal or postnatal survival, growth, or development of the F1 offspring .
2.6.6.1.1. Test Article
The BNT162b2 vaccine is a preservative -free, sterile dispersion of RNA formula ted in L NP
in aque ous cry oprotectant buffer for IM administration.
BioNTech has developed anRNA vaccine platform which utilizes nucleoside -modified
mRNA (modRNA) with blunted innate immune activating capacit y and augmented antigen
expression. The semodRNA -based vaccines ar e formulated in LNPs and encode the
SARS -CoV -2 P2 mutant S glycoprotein (P2 S). Each candidate is also given a V number that
indicates the specific version of the optimized insert genomic sequence but still coding for
the same antigen . The 2 related varian ts of BNT162b2 evaluated in the repeat -dose toxicity
studies are described below:
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Page 8BNT162b2 (V9) (RBP020.2): modRNA encoding the SARS -CoV -2 full -length, P2
mutant, prefusion -stabilized spike gl ycoprotein (P2 S) (V9) -final candidate
BNT162b2 (V8) ( RBP020.1 ): modRNA encoding the SARS -CoV -2 full -length, P2
mutant, prefusion -stabilized spike gl ycoprotein (P2 S) (V8) –related variant
Doses up to 100 µg RNA/dose of the BNT162b2 vaccine candidate have been evaluated in
the clinic . The dose of BNT162b2 (V9) selec ted for licensure is 30 µg RNA/dose.
Each vaccine isformulated in an LNP containing 4 lipids: ALC -0315 , ALC-0159 , DSPC ,
and cholesterol .Other excipients in the formulation include sucrose, NaCl, KC l, Na2HPO 4,
and KH 2PO 4.
Either saline or a solution of phosphate -buffered saline with 300 mM sucrose was used to
dose animals that received buffer control.
IM administration was chosen as this is the clinical route of administration . Doses of
BNT162b2 (V8 or V9) were administered in the nonclinical safet y studies as one 60 or two
100 µ L injections/dosing day (at 30 or 100 µg RNA , respectively ) into the left and/or right
quadriceps muscles. RNA concentrations for the BNT162b2 (V8 and V9) batches used in the
repeat -dose toxicity and DART studies were approximately 0.5mg/mL.
2.6.6.1.2. Animals
Rats were selected asthespecies for assessing the toxicity of the BNT162b2 vaccine as the y
demonstrated an immune response to the BNT162b2 vaccine antigen (Section 2.6.2. 7) and
are a commonly used species in toxicity studies with a large historical database .
Wistar Han rats supplied by Charles River Laboratories ( German y) GmbH were used in the
repeat -dose toxicity study (Study 38166 ) with BNT162b2 (V8). Wistar Han rats supplied by
Charles River Labo ratories (USA) were used in the repeat- dose toxicity study
(Study 20GR142) with BNT162 (V9). Wistar Han rats supplied by Charles River
Laboratories (France) were used in the combined fertility and developmental study
(Study 20256434 ).
2.6.6.2. Single -Dose Toxicity
A separate single -dose toxicity study with the BNT162b2 vaccine candidate has not been
conducted.
2.6.6.3. Repeat -Dose Toxicity
2.6.6.3.1. Repeat -Dose Toxicity Study of Three LNP -Formulated RNA Platforms
Encoding for Viral Proteins by Repeated Intramuscular Administration to Wistar Han
Rats
The objective of this pivotal repeat- dose toxicity study was to determine the potential toxicity
of three LNP --formulated RNA vaccine platforms ,encoding SARS -CoV -2 P2 S or RBD,
administered once weekly b y IMadministration to rats and to a ssess the reversibility of an y
effect safter a 3- week recovery phase (Study 38166 ;Tabulated Summary 2.6.7.7A ).The LNP
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Page 9formulation was the same for the three RNA platforms administered in this study . As the
vaccine candidate selected for licensure is BNT162b2 (V9) , the summary of the results
described below will focus only on the closel y related variant, BNT162b2 (V8), which was
evaluated in this study . However, overall findings were similar among the vaccine candidates
evaluated with the 3 RNA platfor ms. Details on the findings with the other vaccine
candidates evaluated can be found in the study repor t.
Wistar Han rats (15/sex/group) were administered doses of 0 (buffer) or 100µg
RNA/dose /animal BNT162b2 (V8) via IM injection . Doses were administered once a week
for 3 weeks (Day s1, 8, 15). The d ose volume was 200µL/dosing day (100 µLinjected into
each hindlimb) . Following the dosing phase , 10animals/sex from each group were
euthanized 2 days post the last immunization for post- mortem assessments. The remaining
5 animals/sex/group were euthanized following a 3- week recovery phase . Additional satellite
animals (3/sex/group) were used for blood sampling for cy tokine anal ysis.
Clinical signs of toxicity were assessed twice daily throughout the study . Body weights were
recorded twice weekl y during the dosing and the recovery phase. Food consumption was
evaluated once weekl y. Local tolerance (injection site dermal assessment) was evaluated
after each administration, and body temperatures were evaluated at 4 and 24 hours after each
administration. Serum cytokines (IFN -γ, TNF -α, IL-1β, IL -6, IL -10) were evaluated prior to
and 6 hours post each dose and at the end of the dosing phase. Clinical pathology
(hematology and clinical chemistry parameters as well as acute phase proteins) was evaluated
3days after the first administration and at the end of the dosing and recovery phases.
Urinalysis, coagulation parameters, auditory and ophthalmological parameters, serology ,
organ weights, macroscopic and microscopic pathology were evaluated at the end of dosing
and recovery phases.
IM a dministration of BNT162b2 (V8) once weekly for 3 administrations to male and female
Wistar Han rats was tolerated without evidence of sy stemic toxicity and produced the
expected local i nflammatory reaction.
No test article -related unscheduled euthanasias or deaths occurred during the study . There
were no test -item related ophthalmologic or auditory alterations exhibited . There were also
no test article -related s ystemic changes in behavio r, external appearance, or consistency of
feces .
Clinical findings included transient decreases in mean bod y weight and transient increases in
mean bod y temperature. The mean bod y weight of the BTN162b2 (V8) group was transientl y
decreased after each admin istration compared with predose values (down to 0.92x) but were
close to comparable to controls by the end of recovery . The mean body temperature of the
BTN162b2 (V8) group was transiently higher at 4 and/or 24 hours after each administration
compared with the control group. There were no test item effects on body weight or body
temperature during the recovery phase .
Test article -related injection site observations included edema anderythema; with edema
being the most common finding. After the first administration, most animals (23 of 30)
administered BNT162b2 (V8) developed very slight edema or rarel y, slight erythema . The
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Page 10incidence of injection site observations was higher and the observations were more severe
(up to moderate edema ormore rarel y severe edema or ery thema ) after the second and third
dose administration compared with the first administration. However, all observations
resolved prior to the subsequent dosing and were fully recovered at the end of the 3-week
recovery phase. The occurrence of higher severity local reactions after boost immunizations
was attributed to the short immunization interval andto the high vaccine dose, in relation to
the body weight of the rat (approximately up to 0.5 mg/kg ). Macroscopic findings at the
injection sites included induration or thickening, which was noted for 16 of 20 BNT162b2
(V8) -treated animals at the end of the dosing phase . This correlated microscopicall ywith
mild to marked inflammation in all BNT162b2 (V8) -administered animals at the end of the
dosing phase . Inflammation was mixed to mononuclear (characterized by infiltrates of
macrophages, granulocy tes, and ly mphocy tes into the muscle, and variabl y into the dermis
and subcutis) with fibrosis, minimal to marked edema, and minimal to mild myofiber
degeneration (very rarely,minimal necrosis). Inflammation was occasionally evident
extending into tissues adjacent to the injection site (including perineural tissue of sciatic
nerve, tissue around the femur/knee and to the draining [iliac] lymph node )and was
accompanied b y elevations in circulating WBC (up to 2.2x controls), NEUT (up to 7.8x
controls), EOS (up to 6.1x controls), BASO (up to 2.5x controls), and L UC (up to 7.7x
controls) and acute phase proteins (fibrinogen [up to 3.1x controls] , alpha -2-macroglobulin
[up to 217xof controls] , and alpha -1-acid gl ycoprotein [up to 21xof controls] ). Consistent
with an acute phase response (Sellers et al, 2020 ), lower plasma albumin (down to 0.87x
control s) and higher plasma globulin (up to 1.2x controls), resulting in an altered A:G ratio,
were observed in BNT162b2 (V8) -dosed animals. The findings were ty pical of an
inflammatory response to L NP-encapsulated mRNA vaccines. The injection site findings
were not interpreted as adverse because of lack of sy stemic toxicity and absence of clinical
signs of lameness.
Effects considered secondary to immune activation/acute phase responses and inflammation
at the injection site included transient lower RETIC (down t o 0.28x controls; D ay4 onl y),
minimal lower red cell mass parameters (RBC, HGB, and HCT ; down to 0.87x controls ) on
Day17 only , and sporadic lower PLT (down to 0.66x controls) , which were small in
magnitude . PLTreductions were likel y due to inflammation -related PL Tactivation and
consumption and were unassociated with alterations in hemostasis.
At the end of the 3 -week recovery phase, all clinical injection site findings, clinical pathology
findings ,and macrosc opic observations described above had resolved and there was evidence
of recovery of the injection site inflammation microscopicall y.
Test article -related macroscopic enlargement of the draining (iliac) l ymph nodes was evident
at the end of dosing. Microsc opicall y, this finding correlated with mild to moderate increased
cellularity of germinal centers and mild to moderate increased plasma cells in the draining
(iliac) l ymph node and is an anticipated immune response to the administered vaccine and
LNP. At t he end of the 3 -week recovery phase , a few animals administered BNT162b2 (V8)
still had slightly enlarged iliac ly mph nodes. All other BNT162b2 (V8) -related changes in the
draining l ymph node had resolved .
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Page 11Test article -related macroscopic enlargement of sp leen and associated absolute and relative
(to body weight) spleen weights (upto 1.7xcontrols) correlated microscopically to minimal
to mild increased hematopoiesis. Minimal i ncreased hematopoiesis was also evident in the
bone marrow. Both findings were fully resolved at the end of the 3- week recovery phase .
Test article -related microscopic vacuolation of portal hepatocy tes (minimal to mild) was
present in most animals (19 of 20) administered BNT162 b2(V8) at the end of the dosing
phase . This finding was not adverse because it was unassociated with alterations in hepatic
function (eg, no elevations in ALTor AST ) and was fully reversed at the end of the 3 -week
recovery phase .This change may be relat ed to hepatic distribution of the lipids from the LNP
(Sedic etal,2018 ).
Higher GGT (up to 4.6x controls) , which is a biomarker of biliary , not hepatocellular injury
(Boon eetal, 2005 ),was evident in all BNT162b2 (V8) -administer ed animals on Day s 4
and/or 17. There were no other hepatobiliary biomarker alterations or macroscopic or
microscopic findi ngs consistent with cholestasis or hepatobiliary injury to explain the higher
level of GGT, which was completely resolved at the end of the 3- week recovery phase .
No BNT162b2 (V8) -related changes were observed for cy tokine serum concentrations o r in
urinal ysis parameters .
Immunogenicit y assessment demonstrated that BNT162 b2(V8) elicited a SARS -CoV -2 S
-binding IgGresponse directed against the S1 fragment and the RBD. Antibody responses
detected via ELISA correlated with neutralizing activity as seen in th e pseudovirus
neutralization test with BNT162 b2(V8) eliciting higher antigen -binding IgG levels and also
higher pseudovirus neutralization titers. Further details can be found in Section 2.6.2.7.
In conclusion, administration of BNT162b2 (V8) via IMinjections once weekl y for
3 administrations to male and female Wistar Han rats was tolerated without evidence of
systemic toxicity , elicited a robust antigen- specific immune response, and produced
nonadverse inflammatory changes at the injection sites an d the draining lymph nodes,
increased hematopoiesis in the bone marrow and spleen, and clinical pathology changes
consistent with an immune response or inflammation atthe injection sites. There was
nonadverse minimal hepatocellular vacuolation in periport al regions of the liver tha tmay be
related to hepatic distribution of the lipid in the LNP. The findings in this study were
nonadverse, reversible ,andconsistent with those ty pically associated with the IM
administration of LNP-encapsulated mRNA vaccines (Hassett et al, 2019 ).
2.6.6.3.2. 17 -Day Intramuscular Toxicity Study of BNT162b2 (V9) i n Wistar Han Rats
With a 3- Week Recovery
The objectives of this pivotal repeat -dose toxicity study were to determine the potential
toxicity and development of a specific immune response to the antigens in each of the
vaccine candidates, BNT162b2 (V9)and BNT162b3c, administered once weekl y byIM
injection for a total of 3 doses to Wistar Han rats ( Study 20GR142 ; Tabulated
Summary 2.6.7.7B ).The reversibility of potential effects were evaluated following a 3 -week
recovery phase. As the vaccine candidate selected for licensure w as BNT162b2 (V9) , the
summary of the results described below will focus on only that candidate. However, overall
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Page 12findings were similar between the two candidates. Details on the findings with the other
vaccine candidate evaluated in this study , BNT162b3 c, can be found in the study report.
Wistar Han rats (15/sex/group) were administered IM doses of 0 (saline) or 30 µg
RNA/dose /animal BNT162b2 (V9). Doses were administered once a week for 3 weeks
(Day s 1, 8, 15) at a dose volume of 60 µL/dose. Following the dosing phase , 10animals/sex
from each group were euthanized 2 days post last immunization for post -mortem
assessments. The remaining 5 animals/sex/group were euthanized following a 3 -week
recovery phase .
Clinical signs were assessed twice dail y throughout the study . Bod y weights were recorded
twice prior to the initiation of dosing, predose on Day s1, 8, and 15, and on Day s4 and 11,
twice weekly during the recovery phase and just prior to scheduled necrop sy.Food
consumption was evaluated on Day s 4, 8, 11 ,and 15 and twice weekl y during the recovery
phase. Local tolerance (injection site dermal assessment) was evaluated 4 and 24hours after
each administration and at 72 hours post -last dose for recovery animals. Addition al injection
site assessments 48 and 72 hours post injection were collected for animals that had a score of
2 or greater at 24 hours. B ody temperature measurements were taken predose on Day s 1, 8,
and 15 and again at 4 and 24 hours postdose. Clinical pathology (hematology , clinical
chemistry parameters, as well as acute phase proteins) was evaluated on Day s4 and 17 and at
the end of the recovery phase. Urinal ysis, coagulation parameters, and ophthalmological
parameters, serology , organ weigh ts, macroscopic and microscopic pathology were evaluated
at the end of dosing and recovery phases.
There was no unscheduled euthanasia. All animals administered BNT162b2 (V9) survived
to scheduled necropsy at the end of the dosing or recovery phase of the study . There were no
vaccine -related clinical signs observed , or changes to urinaly sis or ophthalmoscopic
parameters during the dosing phase of the stud y.
Test article -related lower mean food consumption ( down to 0.83x control s) was noted on
Days4 and 11 for animals receiving BNT162b2 (V9) .Test article -related higher mean food
consumption (1.08x- 1.35x control) was noted throughout the recovery phase for male
animals administered BNT162b2 (V9 ). No test article -related mean bod y weight changes
were noted for animals administered BNT162b2 (V9) during the dosing phase. Test article -
related higher mean bod y weight (1. 05x-1.06x control) was noted in males only on Recovery
Days 11, 15, 18, and 21 for animals administered BNT162b2 (V9).
Test article related higher mean body temperature (maximum increase post each dose)
compared with concurrent control was noted on Day s1 (up to 0.54°C), 8 ( up to 0.98°C) and
15 (up to 1.03°C) post dose administration o f BNT162b2 (V9). No animal had a bod y
temperature above 40°C through the dosing phase of the study .
BNT162b2 (V9) -related injection site edema Grade 2 (slight, edges of area well defined by
definite raising) or Grade 3 (moderate, raised approximately 1 mm) were noted in most
animals and occurred following dosing on Days 1, 8 and/or 15. The edema was generall y
observed up to 72 hours postdose and fully resolve d.Erythema was also observed at the
injection site in most animals following each dose administration ;however, it was only a
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Page 13Grade 1 (very slight, barely perceptible) and full y resolved prior to the next dose
administration and after the last administration.
BNT162b2 (V9) -related changes in clinical pathology parameters included higher WBC and
fibrinog enand lower A:G ratios, RETI C, RBC mass parameters. H igher WBC ( upto 2.64x
controls), primaril y involving NEUT (up to 6.60x controls), MONO (up to 3.30x controls ),
and LUC (up to 13.2x controls) but also affecting EOS (up to 3.17x controls) and BASO (up
to 8.00x controls) were present on Day s 4 and17, with higher values on Day 17. Lower A:G
ratios ( down to 0.82x controls; with associated but more variable lower total proteins and
albumin [down to 0.92x and 0.85x controls , respectively ] and/or higher globulin [up to 1.10x
controls]) were observed on Day s4 and 17. Hig herfibrinogen occurred on Day 17 (up to
2.49x controls ), consistent with an acute phase response. The acute phase proteins
alpha -1-acid gl ycoprotein (up to 39x controls on Day 17) and alpha -2 macroglobulin (up to
71x controls on Day 17) were elevated in both males and females in the BNT162b2 (V9) -
administered group on Days 4and 17 with higher concentrations generally observed in
males. Transientl y lower RETI C were present on Day 4 (down to 0.27x controls) and higher
RETI C were present on Day 17 (1.31x controls; females only ). Lower RBC mass parameters
(RBC , HGB , HCT; up to 0.90x controls) were present on Day s 4 and17.All test article -
related clinical pathology changes noted in t he dosing phase were full y reversed after a
3-week recovery phase, with the exception of higher red cell distribution width, higher
globulins ,and lower A:G ratio (females) administered BNT162b2(V9).
There were test article -related higher spleen weights, macroscopic observations of enlarged
draining (iliac) ly mph node s,anddiscolored or firm injection sites. Test article -related higher
group mean absolute and relative (to body and brain weight) spleen weights were present in
males (up to1.42x controls) and females (upto1.62x controls) administered BNT162b2
(V9) . Test article -related macroscopic findings included the observation of large draining
lymph nodes (abnormal size, enlarged ; 1of 10 males and 1 of 10 females ) and pale/dark or
firm injection sites (abnormal color, dark/pale and abnormal consistency , 2of 10males and 3
of 10females; firm, 2of 10 males and 4 of 10 females ) in animals administered
BNT162b 2(V9) .At the end of recovery , no test article -related organ weight changes were
noted and macroscopic findings were limited to large draining l ymph nodes (abnormal size,
enlarged) i ndicating a partial recovery of these findings. Pale/dark and/or firm injection sites
and enlarged spleen were not observed at the end of recovery phase indicati ng a complete
recovery of these findings.
At the conclusion of the dosing phase, nonadverse test article -related microscopic findings
consistent with immune activation and an inflammatory response included mixed cell
inflammation and edema of the injection sites (which correlated with macroscopic
observations of abnormal color, dark/pale and abnormal consistency , firm), increased
cellularity of plasma cells and germinal centers of the draining (iliac) and inguinal l ymph
nodes (which correlated with macrosco pic observation of abnormal size of iliac , enlarge d),
increased cellularit y of hematopoietic cells and germinal centers of the spleen (which
correlated with macroscopic observation of abnormal size, enlarged and increased spleen
weights), and increased cel lularity of hematopoietic cells in the bone marrow were noted.
These test article -related changes full y recovered, except for partial recovery of enlarged
draining (iliac) lymph nodes and microscopic findings of inflammation at the injection sites,
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Page 14increas ed cellularit y of plasma cells and germinal centers in the draining and inguinal l ymph
nodes and increased cellularity of the germinal centers in the spleen.
In addition, test article -related vacuolation of the periportal hepatocy tes in the liver was
obser ved, in the absence of biochemical evidence of liver injury , and may be related to
hepatic distribution of LNP lipids (Sedic et al, 2018) . At the end of the 3 -week recovery
phase, thi s finding was completely recovered.
Administration of 3 once weekl y doses of BNT162b2 (V9) elicited SARS -CoV -2
neutralizing antibod y responses in both males and females at the end of the dosing and
recovery phases of the study . SARS -CoV -2 neutralizing anti body responses were not
observed in animals prior to vaccine administration or in saline -administered control animals.
In conclusion, BNT162b2 (V9) administered via IMinjection once weekly for a total of 3
doses to Wistar Han (Crl:WI [Han]) rats was tolera ted without evidence of systemic toxicity ,
generated a SARS -CoV -2 neutralizing antibod y response, and produced nonadverse changes
consistent with an immune or inflammatory response at the conclusion of the dosing phase.
At the end of the 3 -week recovery phase, full or partial recovery of all findings was observed.
Other nonadverse findings included vacuolation in the liver which may be related to hepatic
distribution of L NPlipids and was noted at the conclusion of the dosing phase and
completely recovered. The findings in this study are consistent with those ty picall y
associated with the IM administration of L NP-encapsulated mRNA vaccines. Animals
administered BNT162b2 (V9) elicited SARS- CoV -2 neutralizing antibod y responses at the
end of the dosing and rec overy phases of the study .
2.6.6.4. Genotoxicity
No genotoxicity studies are planned for BNT162b2 ,as the components of all vaccine
constructs are lipids and RNA that are not expected to have genotoxic potential
(WHO,2005 ).
2.6.6.5. Carcinogenicity
Carcinogenicit y studies with BNT162b2 have not been conducted as the components of all
vaccine constructs are lipids and RNA that are not expected to have carci nogenic or
tumorigenic potential . Carcinogenicit y testing is generall y not considered necessary to
support the development and licensure of vaccine products for infectious diseases
(WHO, 2005 ).
2.6.6.6. Reproductive and Developmental Toxicity
Overall, there were no effects of BNT162b2 administration on female fertility , pregnancy , or
embr yo-fetal or offspring development. In addition, m acroscopic and microscopic evaluation
of male and female reproductive tissues from the repeat -dose toxicity studies with
BNT162b2 sh owed no evidence of toxicity .
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Page 152.6.6.6.1. A Combined Fertility and Developmental Study (Including Teratogenicity and
Postnatal Investigations) of BNT162b1, BNT162b2 and BNT162b3 by Intramuscular
Administration in the Wistar Han Rat
BNT162b2 (V9) was administered by IMinjection at the human clinical dose ( 30µg
RNA /dosing day ) to 44 female Wistar Han ra ts (F0) 21 and 14days prior to mating with
untreated males and on GDs 9 and 2 0, for a total of 4 dosing day s (Study 20256434 ). A
separate control group of 44 F0females received saline by the same route and regimen. This
study also included assessment of two other LNP -formulated RNA vaccine candidate s
(BNT162b1 and BNT162b3) that did not proceed into Phase 2/3 clinical trials. Here , the
study findings from BNT162 b2are summarized; findings from the BNT162b1 and
BNT162b3 vaccine candidates also tested in this study were generally similar and can be
found in the study report.
Following completion of a mating phase with untreated males, 22 rats/group underwent
caesar ean-section on GD 2 1 and were submitted to routine embry o-fetal development
evaluations. The remaining 22 rats/group were allowed to litter and behavior of the mothers
and development of the offspring was observed until PND 21.
There were no BNT162b2 -related deaths during the study .IMadministration of BNT162b2
before and during gestation to female Wistar rats resulted in non-adverse clinical signs and
macroscopic findings localized to the injection site as well as transient, non -adverse bod y
weight and food consumption effects after each dose administration. These maternal findings
are all consistent with administration of a vaccine and an inflammatory /immune response and
with those observed in the repeat -dose toxicity stud ieswith BNT162b2.
There were no BNT162b2 -related effects on an y mating or fertility parameters. There were
no BNT162b2 -related effects on any ovarian, uterine, or litter parameters, including embryo -
fetal survival, growth, or external, visceral, or skeletal malformations, anomalies, or
variations. There were no effects of BNT162b2 administration on postnatal offspring (F1)
development, including postnatal growth, phy sical development (pinna unfolding and ey e
opening), neurodevelopment (pre -weaning auditory and visual function tests), macr oscopic
observations, and survival.
All of F0 females administered BNT162b2 developed a SARS -CoV -2 neutralizing antibody
response and these responses were detectable in all fetuses and pups from the caesarean and
littering groups, respectively . The animals in the saline control group did not exhibit an
immune response to BNT162b2 .
In conclusion, administration of BNT162b2 (V9) to female rats twice before the start of
mating a nd twice during gestation at the human clinical dose was associated with non -
adverse effects (bod y weight, food consumption and effects localized to the injection site)
after each dose administration. However, there were no effects of BNT162b2 administrati on
on mating performance, fertility , or an y ovarian or uterine parameters in the F0 female rats
nor on embry o-fetal or postnatal survival, growth, or development in the F1 offspring. An
immune response was confirmed in F0 female rats following administrati on of BNT162b2
and these responses were also detectable in the F1 offspring (fetuses and pups).
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Page 162.6.6.7. Local Tolerance
Local tolerance of IM administration of BNT162b2 was evaluated by injection site
observations and macroscopic and microscopic examination of inj ection sites in the pivotal
repeat -dose toxicity studies and are described above ( Section 2.6.6.3 ).
2.6.6.8. Other Toxicity Studies (if availa ble)
2.6.6.8.1. Antigenicity
Immunogenicit y was evaluated as part of the primary pharmacology studies ( Sections 2.6.2.5
and 2.6.2.6 ). In general, administration of BNT162b2 variants (V8 and V9) generated a
robust immune response in non- GLP mouse and NHP immunogenicit y studies. Serology data
from the repeat -dose toxicity studies and the DART study showed a robust antigen -specific
immune response to BNT162b2 (2.6.2. 7).
2.6.6.8.2. Immunotoxicity
Stand -alone immunotoxicity studies with BNT162b2 have not been conducted. However,
immunotoxicological endpoints have been collected as part of the pivotal repeat -dose toxicity
studies. There were no adverse effects observed and no significant effects on measured
cytokines.
2.6.6.9. Discussion and Conclusions
Administration of BNT162b2 by IM injection to male and female Wistar Han rats once every
week for a total of 3 weekly cycles of dosing was tolerated without evidence of s ystemic
toxicity in GL P-compliant repeat -dose toxicity studies. Expected inflammatory responses to
the vaccine we re evident such as edema and ery thema at the injection sites, transient
elevation in body temperature, elevations in WBCs and acute phase reactants, and lower A:G
ratios. A transient elevation in GGT was noted in animals administered BNT162b2 (V8)
inStudy 38166 without evidence of microscopic changes in the biliary system or other
hepatobiliary biomarkers but was not recapitulated in Study 20GR142. Injection site
reactions were common in all vaccine -administered animals and were greater after boost
immuniz ations. Changes secondary to inflammation included slight and transient reduction in
body weights and transient reduction in RETIC, PLT, and RBC mass parameters. All
changes in clinical pathology parameters and acute phase proteins were reversed at the end
ofthe recovery phase for BNT162b2 with the exception of higher red cell distribution width,
higher globulins, and lower A:G ratios in animals administered BNT162b2 (V9).
Macroscopic pathology and organ weight changes were also consistent with immune
activation and inflammatory response and included increased size of draining iliac l ymph
nodes and increased size and weight of spleen. Vaccine-related microscopic findings at the
end of the dosing phase consisted of edema and inflammation in injection sites a nd
surrounding tissue; increased cellularity in the draining iliac and inguinal lymph nodes, bone
marrow, and spleen; and hepatocy te vacuolation in the liver. P eriportal vacuolation of
hepatocy tes was not associated with any microscopic evidence of hepatic injury or alterations
in liver function tests and is interpreted to reflect hepatocy te uptake of the LNP lipids
(Sedic et al, 2018). Microscopic findings at the end of the dosing phase were partiall y or
completely recovered in all animals at the end of the recovery phase for BNT162b2. A robust
immune response was elicited to the BNT162b2 ant igen.
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Page 17Administration of BNT162b2 (V9) to female rats twice prior to mating and twice during
gestation resulted in maternal observations (local reactions, transient decreases in body
weight and food consumption) similar to those seen in the repeat -dose toxi city studies.
However, there were no BNT162b2 -related effects on female fertility , pregnancy , or embry o-
fetal or offspring development in the presence of SARS- CoV -2 neutralizing antibodies in the
maternal animals, fetuses, and pups. This is consistent with the observation of no
macroscopic or microscopic findings in reproductive organs in the repeat -dose toxicity
studies .
The results of the rat repeat -dose toxicity studies with the BNT162b2 variants ( V8 and V9)
and DART study with BNT162b2 (V9) demonstrate tolerability of the COVID -19vaccine .
Given the lack of adverse findings in the rats related to COVI D-19 vaccine administration,
the nonclinical toxicity program supports the clinical administration of BNT162b2 twice by
IM injection at a dose of 30 µgRNA .
2.6.6.10. References
Boone L, Mey er D, Cusick P, et al. Selection and interpretation of clinical pathology
indicators of hepatic injury in preclinical studies. Vet Clin Pathol 2005;34(3):182 -8.
Hassett KJ, Benenato KE, Jacquinet E, et al. Optimization of lipid nano particles for
intramuscular administration of mRNA vaccines. Mol Ther Nucleic Acids 2019;15:1 -11.
Sedic M,Senn J, Ly nn A, et al. Safet y Evaluation of L ipid Nanoparticle –Formulated
Modified mRNA in the Sprague -Dawley Rat and Cy nomolgus Monkey . Vet
Path 2018;55(2):341 -54.
Sellers RS, Nelson K, Bindu B, et al. Scientific and Regulatory Policy Committee Points to
Consider: Approaches to the Conduct and Interpretation of Vaccine Safet y Studies for
Clinical and Anatomic Pathologists. Toxicol Pathol 2020;48(2):257 -76.
US Department of Health and Human Services, Food and Drug Administration, Center for
Biologics Evaluation and Research. Development and licensure of vaccines to prevent
COVID -19. In: Guidance for industry . Rockville, MD: Food and Drug Administration; 2020:
21 pages.
World Health Organization. WHO guidelines on nonclinical evaluation of vaccines. Annex 1.
In: World Health Organization. WHO technical report series, no. 927. Geneva, Switzerland;
World Health Organization; 2005:31 -63.
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