Methanal, reaction products with 1,3-bis(aminomethyl)benzene and hydroxybenzene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-[3-(aminomethyl)phenyl]methanamine; 2,4-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; 2,6-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; phenol

EC Number:

701-207-5

Cas Number:

1950616-36-0

Molecular formula:

(C8 H12 N2 . C6 H6 O . C H2 O)x

IUPAC Name:

1-[3-(aminomethyl)phenyl]methanamine; 2,4-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; 2,6-bis[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]-4-[(4-hydroxyphenyl)methyl]phenol; 2-[({[3-(aminomethyl)phenyl]methyl}amino)methyl]phenol; phenol

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

Animal Information
Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals 88 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Five days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 70 to 76 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 232 to 295 g.

Mating
Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating

Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals
were not part of the study and were maintained as stock animals.
Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method To group and cage position in the sequence of mating.
Females mating on any one day were evenly distributed amongst the groups.
Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.
Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24C and
40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Acclimatization up to four animals
During pairing one (stock) male and one female
Gestation one female

Environmental Enrichment
Aspen wood based products A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified, pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was
released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free
fiber bedding and Aspen wood based products.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Oral, by gavage, using a suitably graduated syringe and a flexible cannula inserted via the mouth.
As a precautionary measure to reduce the risk of any potential irritation to the mouth and/or esophagus during gavage administration, the dose was drawn up via the cannula and the external surface of the cannula was wiped on paper tissue. The canula surface was rinsed in a container of tap water (container 1) and wiped dry on clean paper tissue. Then the outside of the cannula was rinsed further in a second container of tap water (container 2) prior to intubation. Fresh containers of tap water were used for each group.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity
The homogeneity and stability of formulations during storage were confirmed as part of another study, Covance Study Number CS73HX. Stability was confirmed to be stable for four days refrigerated (2 to 8°C) and for one day at ambient temperature (15 to 25°C).

Achieved concentration
Samples of each formulation prepared for administration in the first (Group 4 at 60 mg/mL) and last (Group 4 at 40 mg/mL) preparation were analyzed for achieved concentration of the test item. Samples were also analyzed for achieved concentration of the test item on 02 February 2021.

Details on mating procedure:

Male/female ratio 1:1 with identified stock males.
Daily checks for evidence of mating

Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
Day 0 of gestation When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 female/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Purpose
The purpose of this study was to assess the influence of Methanal, reaction products with 1.3-
bis(aminomethyl)benzene and hydroxybenzen (an industrial chemical), on embryo-fetal
survival and development when administered during the organogenesis and fetal growth
phases of pregnancy (Days 6 to 19 after mating) in the Sprague-Dawley rat.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the
historical control data available at this laboratory.

Route of Administration
The oral gavage route of administration was chosen as administration by gavage is a common
and accepted route of exposure for studies of this type.

Rationale for Dose Level Selection
The doses used in this study (0, 30, 100 or 300/200 mg/kg/day) were selected in conjunction
with the Sponsor based on the findings of a preliminary Embryo-Fetal Study (Covance Study
Number: 8428167).
In the preliminary study dose levels of 250, 500 or 750 mg/kg/day were investigated. There
were four deaths that were related to treatment with the test item. These deaths comprised:
three females given 750 mg/kg/day and one female given 500 mg/kg/day. The breathing of
all these animals was impaired which necessitated their premature termination. Macroscopic
examination indicated disturbance of the gastro intestinal tract in all animals; additionally at
750 mg/kg/day depressions in the glandular mucosa of the stomach were also apparent for
one animal and the nasal turbinates could not be flushed for another animal. In view of these
deaths at 750 mg/kg/day, a decision was taken to terminate this dose group and all surviving
females were prematurely terminated. Treatment at 750 or 500 mg/kg/day was also
associated with low body weight gain and food consumption. Treatment at 250 mg/kg/day
was generally well tolerated, although two females did show rales during the study, and there
were no premature deaths throughout the duration of the study.
Due to the toxicity at 500 or 750 mg/kg/day, these dose levels were not considered suitable
for use on this embryo-fetal study. A high dose level of 300 mg/kg/day was selected for use
on this study. Low and intermediate dose levels of 30 and 100 mg/kg/day were selected to
provide an approximate three-fold interval between dose level.

Examinations

Maternal examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases as described in Section 3.7.

Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to
treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupant(s). Any deviation from normal was recorded at the time in respect of nature and
severity, date and time of onset, duration and progress of the observed condition, as
appropriate.
During the acclimatization period, observations of the animals and their cages were recorded
at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times
in relation to dose administration:
 Pre-dose observation
 One to two hours after completion of dosing
 As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20
after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was
recorded for the periods Days 0-2, 3-5, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating
inclusive.

Thyroid Hormone Analysis
Blood samples were collected at the following occasion:
Occasion Animals
At scheduled termination All adults (excluding premature deaths)
Sequence of blood sampling on each occasion

To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.

Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Conditions No overnight deprivation of food.
Blood sample site Sublingual vein.
Anesthetic Isoflurane.
Anticoagulant None.
Tubes Greiner Minicollect tubes with clotting activator.
Blood volume 1.0 mL.
Treatment of samples Samples were kept at ambient temperature (15 to 25°C) for a
minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
All available serum was transferred to appropriately labelled
polypropylene “cryo” tubes using a plastic disposable pipette,
and then mixed by gentle 10-fold inversion. Following
mixing, each serum sample was divided in two aliquots.
Number of aliquots Two per animal. Aliquot 1: 0.2 mL serum for T3/T4
Aliquot 2: residual serum for TSH
Final storage conditions Deep frozen (approximately -60°C to -90C) pending
analysis.
Fate of samples Samples were dispatched on dry ice to the Huntingdon site to the Contributing Scientists responsible for analysis.
T3 and T4 Performed by the Department of Bioanalysis, Covance.
The method of analysis and results are presented in
Attachment 14.3.
TSH Performed by the Department of Immunology and
Immunotoxicology, Covance.
The method of analysis and results are presented in
Attachment 14.4

Terminal Investigations
Method of Kill
Method of kill for all adult
animals

Carbon dioxide asphyxiation.

Method of kill for fetuses Chilling on a cool plate (approximately 0C).

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each
animal, a full macroscopic examination of the tissues was performed. All external features
and orifices were examined visually. Any abnormality in the appearance or size of any organ
and tissue (external and cut surface) was recorded and the required tissue samples preserved
in appropriate fixative.
Schedule Animals surviving until the end of the scheduled study
period were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed
as follows:

Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Abnormalities *
Gravid uterine weight
(including cervix and ovaries) *
Thyroid gland *a) * * *
a) Weighed after partial fixation.
* Organs weighed, samples fixed.
Animal ID retained.

Organ Weights
Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Thyroid All adult females.
Routine staining Sections were stained with hematoxylin and eosin.

Light Microscopy
Tissues preserved for examination were examined as follows:

Category Animals Tissues
Premature deaths All animals from all groups. Thyroid gland only
Scheduled kill All animals from all groups. Thyroid gland only

Findings were either reported as "present" or assigned a severity grade. In the latter case one
of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.

Ovaries and uterine content:

For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where
possible):
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

Fetal examinations:

Fetal Examination and Processing
Examination of all viable fetuses and placentae

Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter

Sexed internally and eviscerated

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.

Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.
Examination of all viable fetuses and placentae

Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex and ano-genital distance of each fetus was also recorded.
Fixation Nominally one third of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid.
Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit.
Processing Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses Assessed for skeletal development and abnormalities.

Statistics:

The analyses were carried out using the individual animal as the basic experimental unit. For
litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis
and biological significance was assessed with relevance to the severity of the anomaly and
the incidence of the finding within the background control population.
The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and
sex ratio - percentage male)
Placental, litter and fetal weights
Ano-genital distance, average for each litter adjusted for litter average fetal body weight
Organ weights, absolute
The following comparisons were performed:
Group 1 vs 2, 3 and 4

The following sequence of statistical tests was used for body weight, gravid uterus weight,
food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex
ratio - percentage male, placental, litter and fetal weights, ano-genital distance and organ
weight data:
A parametric analysis was performed if Bartlett's test for variance homogeneity
(Bartlett, 1937) was not significant at the 1% level. For pre-treatment data, analysis of
variance was used to test for any group differences. Where this was significant


Covance Study Number 8437285

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(p<0.05) inter group comparisons using t-tests, with the error mean square from the
one-way analysis of variance, were made. For all other analyses the/The F1
approximate test was applied. This test is designed to detect significant departure
from monotonicity of means when the main test for the comparison of the means is a
parametric monotonic trend test, such as Williams’ test (Williams 1971, 1972). The
test statistic compares the mean square, NMS, for the d

Indices:

Serial Observations
Clinical Observations
Clinical observations are presented for each animal that showed signs, providing detail of the
type of sign, day of occurrence and information on the duration of the sign applicable.
Body Weight
Group mean weight changes were calculated from the weight changes of individual animals.
Body weights were plotted graphically with respect to Day 6 of gestation.


Covance Study Number 8437285

- 23 -

Adjusted body weights on Day 20 after mating were calculated from the body weight at
termination minus the gravid uterine weight. Body weight change values for the period
Day 6-20 were also presented, after being adjusted for the contribution of the gravid uterus.
Food Consumption
Group mean food consumptions and standard deviations for each period were derived from
unrounded cage values. Overall mean food consumption values were calculated for each cage
and the mean of these cage means were calculated for each group.
The column header day numbers represent the days on which the full feeder and empty feeder
were recorded.
3.8.2 Terminal Investigations
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations) x 100
Number of corpora lutea
Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%) = (Number of implantations - Number of live fetuses) x 100
Number of implantations
All group values and SD were calculated from the individual litter values.
Fetal, Litter and Placental Weights
Mean fetal weights were calculated for each litter. Values were presen

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Occasional incidences of intermittent rales (dry/wet) was seen in a few animals receiving
200 mg/kg/day, mainly in the second half of the treatment period. Similar singular
incidences of intermittent rales were apparent for three females receiving 30 mg/kg/day but
this was at a same incidence as observed for the control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

Two females (Numbers 71 and 62) receiving 300 mg/kg/day displayed adverse clinical signs,
resulting in dispatch to necropsy for welfare euthanasia before dosing on Day 7 and 9 of
gestation, after receiving one or two days of treatment, respectively. Signs for both animals
consisted of gasping and irregular breathing, with female 62 also showing piloerection and
rales, which was also present the previous day, and a body weight loss of 25g from the start
of treatment. Macroscopic examination revealed gaseous distension of the gastrointestinal
tract for Female 71 and for Female 62, gas in the jejunum and no contents in the colon or
rectum. Due to the two deaths on study, the decision was taken to reduce the dose level for
the Group 4 animals to 200 mg/kg/day, and after reduction of the dose level there were no
further deaths. Intermittent rales (dry/wet) was seen in a few animals receiving
200 mg/kg/day.
Animal number 46 receiving 100 mg/kg/day was found dead after dosing on Day 18 of
gestation. Macroscopic examination revealed a perforated oesophagus and this death was
considered to reflect accidental trauma during the dosing procedure.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Microscopic findings were restricted to minimal follicular cell hypertrophy in one female
administered 100 mg/kg/day and one female administered 200/300 mg/kg/day. In view of the
low incidence and severity, this finding was not considered test item-related. There were no
other microscopic findings.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 300/200 mg/kg/day, there were four fetuses in one litter with
Ectrodactyly/Polydactyly/Syndactyly fore and/or hind paws, however, as only a single litter
was affected, this was considered a spontaneous occurrence and therefore not treatment
related.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 300/200 mg/kg/day there was slight increase in incidence of abdominal cavity
haemorrhages compared to concurrent control. Although this was outside historical control
data range, it was a minor abnormality and not considered adverse.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

Initial dose levels of Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and
Hydroxybenzen at 30, 100 and 300 mg/kg/day were employed on this main embryo-fetal
study but, shortly after treatment started. it was apparent that treatment at 300 mg/kg/day was
not tolerated, and this dose level was reduced to 200 mg/kg/day during the early
organogenesis period for surviving females.

Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen at dose
levels of 30, 100 or 200 mg/kg/day to Sprague-Dawley rats throughout organogenesis and the
fetal growth phases of pregnancy was well tolerated. At dose levels of 30, 100 or
200 mg/kg/day there was no adverse effect of treatment on clinical condition, body weight
gain or food intake, there were also no treatment-related macroscopic findings, microscopic
findings in the thyroid gland, and embryo-fetal survival and development was not adversely
affected by treatment.

It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal
toxicity and embryo-fetal survival and development is 200 mg/kg/day.

Executive summary:

The purpose of this study was to assess the influence of Methanal, reaction products with 1.3-bis(aminomethyl)benzene and hydroxybenzen (an industrial chemical), on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of pregnancy (Days 6 to 19 after mating) in the Sprague Dawley rat.
Three groups of 20 females received Methanal, reaction products with 1.3-
bis(aminomethyl)benzene and hydroxybenzen at doses of 30, 100 or 300 mg/kg/day by oral gavage administration, from Day 6 to 19 after mating. Following two unexpected deaths which occurred in Group 4; the dose level was reduced from 300 to 200 mg/kg/day effective from 30 January 2021 (Day 8-11 after mating). A similarly constituted Control group received the vehicle, propylene glycol over the same treatment period at the same volume dose as treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.
Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded.
Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.

Results
The mean concentrations of Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen in test formulations analysed from Groups 2, 3 and 4 from the preparation from 02 February 2021 and the Group 4 formulation from the last preparation were within -15% of the nominal concentration, confirming the accuracy of formulation.
However, the analysis of the Group 2 and 3 formulations from the last preparation were -21.2 and -33.5% from nominal respectively.
Two females (Numbers 71 and 62) receiving 300 mg/kg/day displayed adverse clinical signs, resulting in dispatch to necropsy for welfare euthanasia before dosing on Day 7 and 9 of gestation, after receiving one or two days of treatment, respectively. Signs consisted of gasping and irregular breathing, with macroscopic examination showing gaseous distension of part or all of the GI tract. Due to these two deaths the decision was taken to reduce the dose level for the Group 4 animals to 200 mg/kg/day, and following this reduction of the dose level there were no further treatment-related deaths. A slightly higher incidence of intermittent rales (dry/wet) was seen in a few animals receiving 200 mg/kg/day.
Body weight gain throughout treatment, including overall body weight gain when adjusted for the contribution of the gravid uterus, was similar throughout the groups.
Group mean food consumption was similar throughout the groups.
There were no test item-related thyroid gland organ weight changes.
There were no treatment-related macroscopic findings for animals surviving until scheduled termination.

There were no test item-related microscopic findings in the thyroid gland.
The analysis of serum thyroid hormone concentrations performed at scheduled termination revealed, compared with controls, similar mean serum T3, T4 or TSH concentrations in females receiving 30, 100 or 200 mg/kg/day, all of which did not attain statistical
significance.
Litter data, as assessed by the mean numbers of implantations, resorptions (early or late), live young, sex ratio or the levels of pre or post implantation losses was similar in all groups andthere was no indication that maternal treatment had any effect on embryo-fetal survival.
There was no effect of maternal treatment on mean placental, total litter, fetal weights or ano-genital distance.
At detailed fetal pathology examination, the incidence of major and minor abnormalities and skeletal variants show no relationship to treatment.

Conclusion
Initial dose levels of Methanal, reaction products with 1,3- Bis(aminomethyl)benzene and Hydroxybenzen at 30, 100 and 300 mg/kg/day were employed on this main embryo-fetal study but, shortly after treatment started. it was apparent that treatment at 300 mg/kg/day was not tolerated, and this dose level was reduced to 200 mg/kg/day during the early
organogenesis period for surviving females.
Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen at dose levels of 30, 100 or 200 mg/kg/day to Sprague-Dawley rats throughout organogenesis and the fetal growth phases of pregnancy was well tolerated. At dose levels of 30, 100 or 200 mg/kg/day there was no adverse effect of treatment on clinical condition, body weight gain or food intake, there were also no treatment-related macroscopic findings, microscopic findings in the thyroid gland, and embryo-fetal survival and development was not adversely affected by treatment.
It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity and embryo-fetal survival and development is 200 mg/kg/day.