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

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

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

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)

EC Number:

500-607-5

EC Name:

Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)

Cas Number:

161278-17-7

Molecular formula:

unspecified

IUPAC Name:

Formaldehyde, oligomeric reaction products with 4,4'-isopropylidenediphenol and m-phenylenebis (methylamine)

Test material form:

liquid

Specific details on test material used for the study:

Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen
Test item identity (including alternative names)

EPIKURE
EPIKURE 105

CAS number 1950616-36-0
Intended use Industrial chemical, used in many chemical manufacturing processes
Physical state Liquid
Appearance Brown-yellow liquid
Storage conditions At ambient temperature (15 to 25°C), in the dark
Supplier Sponsor
Batch number DG6L50992A
Expiry date 27 January 2022
Purity 100%

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

90 days

Frequency of treatment:

once per day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 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.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

3.6 Serial Observations
3.6.1 Clinical and Behavioral 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
occupants. 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
Daily during the first week of treatment, twice weekly during Weeks 2 to 4 (middle and end
of each week) and weekly thereafter, detailed observations were recorded at the following
times in relation to dose administration
 Pre-dose observation.
 As each animal is returned to home cage.
 At the end of dosing each group.
 One to two hours after completion of dosing of all groups.
 As late as possible in the working day.


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Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, detailed physical
examination and arena observations were performed on each animal. On each occasion, the
examinations were performed at approximately the same time of day (before dosing during
the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and
behavior during handling and after being placed in a standard arena. Any deviation from
normal was recorded with respect to the nature and, where appropriate, degree of severity.
Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor
and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or
marked.
Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all
animals during Week 12 of treatment. Animals were tested by an observer who was unaware
of the treatment group to which each animal belonged. Before the start of observations, cage
labels showing the treatment group were replaced by labels stating only the study, animal and
cage numbers. Animals were not necessarily all tested on the same day, but the numbers of
animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose
(but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction
Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response
Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor


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Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and
the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without
moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with
vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or
aggression
Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges.
Three trials were performed.
At any point during the observations, additional comments were made as free text where
considered appropriate.
Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was
measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware
supplied by Pearson Technical Services and software developed and maintained by Covance.
Animals were tested individually in clear polycarbonate cages and motor activity was
measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten
beams were set at two height levels (five low and five high) to detect cage floor and rearing
activity respectively. Animals were not necessarily all tested on the same day, but the
numbers of animals and the times of testing were balanced across the groups on each day of
testing.
3.6.2 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.
Where possible, blood samples were taken ante mortem and analyzed for the characteristics
specified in the Hematology, peripheral blood and Blood chemistry sections.
A complete necropsy was performed in all cases as described in Section 3.7.
3.6.3 Body Weight
The weight of each animal was recorded three days before treatment commenced, on the day
that treatment commenced (Week 0), weekly throughout the study and before necropsy.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health,
so that the progress of the observed condition could be monitored. These data are retained in
the study data but are not reported.
3.6.4 Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was
recorded for the three days before treatment started and for each week throughout the study.


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3.6.5 Water Consumption
Fluid intake was assessed by daily visual observation. No significant effect was observed and
consequently quantitative measurements were not performed.
3.6.6 Ophthalmic Examination
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as
follows:
Occasion Animals
Pretreatment All animals
Week 13 Groups 1 and 4 only
Prior to each examination, the pupils of each animal were dilated using tropicamide
ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber,
iris (pupil dilated), lens, vitreous and ocular fundus were examined.
3.6.7 Hematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food and prior to dosing at the
following occasion:
Occasion Animals
Week 13 All animals
Sampling was performed on the morning after overnight collection of urine. Animals were,
therefore, also deprived of water overnight but had access to water for a minimum period of
one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. Blood samples
(nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing
EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120
analyzer:
 Hematocrit (Hct)*
 Hemoglobin concentration (Hb)
 Erythrocyte count (RBC)
 Absolute reticulocyte count (Retic)
 Mean cell hemoglobin (MCH)*
 Mean cell hemoglobin concentration (MCHC)*
 Mean cell volume (MCV)
 Red cell distribution width (RDW)
 Total leucocyte count (WBC)
 Differential leucocyte count:
 Neutrophils (N)
 Lymphocytes (L)
 Eosinophils (E)
 Basophils (B)
 Monocytes (M)
 Large unstained cells (LUC)
 Platelet count (Plt)
* Derived values calculated in ClinAxys.


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Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by
light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written
description from the blood film was made where appropriate. A manual count of the
differential white blood cell parameters was performed where necessary.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate
anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate
reagent in respect of
 Prothrombin time (PT) - using IL PT Fibrinogen reagent.
 Activated partial thromboplastin time (APTT) - using IL APTT reagent.
 Clauss fibrinogen concentration (Fib) - using IL PT Fibrinogen reagent.
3.6.8 Blood Chemistry
Blood samples were collected after overnight withdrawal of food and prior to dosing at the
following occasion:
Occasion Animals
Week 13 All animals
Sampling was performed on the morning after overnight collection of urine. Animals were,
therefore, also deprived of water overnight but had access to water for a minimum period of
one hour prior to the commencement of blood sampling procedures.
Animals were held under light general anesthesia induced by isoflurane. Blood samples
(nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes
containing lithium heparin as anticoagulant. After separation, the plasma was examined using
a Roche Cobas 6000 Analyzer in respect of:
 Alkaline phosphatase (ALP)
 Alanine aminotransferase (ALT)
 Aspartate aminotransferase (AST)
 Total bilirubin (Bili)
 Bile acids (BiAc)
 Urea
 Blood urea nitrogen (BUN)
 Creatinine (Creat)
 Glucose (Gluc)
 Total cholesterol (Chol)
 Cholesterol (HDL)
 Cholesterol (LDL)
 Triglycerides (Trig)
 Sodium (Na)
 Potassium (K)
 Chloride (Cl)
 Calcium (Ca)
 Inorganic phosphorus (Phos)
 Total protein (Total Prot)
 Albumin (Alb)


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Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and
analyzed albumin concentration.
3.6.9 Urinalysis
Animals were placed in an individual metabolism cage, without food or water. Urine samples
were collected overnight at the following occasions:
Occasion Animals
Week 13 All animals
The individual samples were examined for the following characteristics:
Using manual methods:
 Clarity and Color (App) - by visual assessment
 Volume (Vol) - using a measuring cylinder
 pH - using a pH meter
 Specific gravity (SG) - by direct refractometry using a SG meter
Using Multistix reagent strips interpreted using the Clinitek500 instrument:
 Ketones (Keto)
 Bile pigments (Bili)
 Blood pigments (UBld)
Using a Roche Cobas 6000 Analyzer:
 Protein - total (T-Prot) and concentration (Prot)
 Creatinine - total (T-Creat) and concentration (U-Creat)
 Glucose - total (T-Gluc) and concentration (U-Gluc)
3.6.10 Estrous cycles
Wet smears Wet smears were taken from the vagina of all females using
pipette lavage for four days before scheduled termination. The
last smear was taken on the morning of necropsy.
Smears were assessed to establish the stage of estrus (metestrus,
diestrus, proestrus and estrus) at termination and were used to
assist in the histological evaluation of estrogen sensitive tissues.
3.6.11 Thyroid Hormone Analysis
Blood samples were collected at necropsy as follows:
Occasion Animals
At termination All animals


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Sequence of blood sampling
on each occasion

In order 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.
Conditions No overnight deprivation of food.
Anesthetic Isoflurane. The animals did not recover from the anesthesia.
Blood sample site Sublingual vein.
Parameters Triiodothyronine (T3)
Thyroxine (T4)
Thyroid stimulating hormone (TSH)
Anticoagulant None.
Blood volume 1.0 mL.
Blood tubes Grenier Minicollect tubes with clotting activator.
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 10 minutes at 4°C.
Aliquot volumes Aliquot 1 (T3 and T4): 0.2 mL of serum
Aliquot 2 (TSH): all remaining serum
Final storage conditions Deep frozen (-60°C to -90C) pending analysis.
Fate of samples Dispatched to the Department of Biomarkers, Bioanalysis and
Clinical Sciences, Covance Huntingdon.
T3/T4 analysis Performed by the Department of Department of LC-
MS/MS Bioanalysis, Covance Huntingdon.
The method of analysis and results are presented in
Attachment 13.3.
TSH analysis Performed by the Department of Immunotoxicology &
Immunoassay, Covance Huntingdon.
The method of analysis and results are presented in
Attachment 13.4.

Sacrifice and pathology:

3.7 Terminal Investigations
3.7.1 Method of Kill
Carbon dioxide asphyxiation with subsequent exsanguination.
3.7.2 Necropsy
All 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


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tissue (external and cut surface) was recorded and the required tissue samples preserved in
appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule Animals were killed following 13 weeks of treatment.
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 * * *
Adrenals * * * *
Aorta * * *
Brain (cerebellum, cerebrum, midbrain) * * * *
Cecum * * *
Colon * * *
Duodenum * * *
Epididymides * L+R * R * R * R
Esophagus * * *
Eyes * # #
Femur (femorotibial joint) *(a) † †
Head * # #
Heart (including auricular and ventricular regions) * * * *
Ileum * * *
Jejunum * * *
Kidneys * * * *
Liver (section from two lobes) * * * *
Lungs (section from two major lobes including bronchi) * * *
Lymph nodes - mesenteric * * *
- left axillary * * *
Ovaries * * * *
Pancreas * * *
Peyer’s patch * * *
Pituitary * * * *
Prostate *(b) * * *
Rectum * * *
Salivary glands - submandibular * † †
- sublingual * † †-
- parotid * † †
Sciatic nerves * † †
Seminal vesicles with coagulating gland *(b) * * *
Skeletal muscle * † †
Skin with mammary glands (inguinal area) * * *
Spinal cord (transverse and longitudinal sections at the
cervical, thoracic and lumbar levels)

* * *
Spleen * * * *
Sternum (and bone marrow) * * *
Stomach * * *
Testes * L+R * R * R * R
Thymus * * * *


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Tissue and regions examined Necropsy Histology Pathology
Weigh Fix Light microscopy
Thyroid with parathyroids *(c) * * *
Trachea * * *
Urinary bladder * * *
Uterus with cervix * * * *
Vagina * * *
Animal ID retained
a Both hind limbs retained, one sectioned where appropriate
b Seminal vesicles and coagulating gland weighted together with prostate
c Weighed after partial fixation
* Organs weighed, samples fixed or sections examined microscopically
# Not examined
† Only one examined
L&R Left and right
Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above.
Requisite organs were weighed for all animals killed at scheduled intervals.
Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of
those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.
3.7.3 Sperm Analysis
Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and
testis was removed and the epididymis and testis were weighed.
The following tests were performed:
Sperm motility - all groups A sample of sperm was expressed from the vas deferens into
pre-warmed (target 37C) medium M199, which contained
0.5% w/v bovine serum albumin (BSA Fraction V). A
sample for assessment was taken into a 100 m depth
cannula by capillary action and at least 200 sperm per
animal analyzed using the Hamilton Thorne IVOS II
Computer Assisted Sperm Analyzer (CASA).
Sperm morphology -
Groups 1 and 4

A 200 L aliquot of the sperm/medium mixture (described
above) was diluted with 800 L of 10% neutral buffered
formalin. After staining with nigrosine and eosin an air-dried
smear was prepared. Slides were examined by light
microscopy for the assessment of sperm morphology. At
least 200 sperm were assessed for each male.
Groups 2 and 3 Fixed samples retained for possible future assessment.


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Sperm count -
Groups 1 and 4

The left cauda epididymis of each male was weighed and
then the tunica was removed then homogenised for at least
30 seconds in 10 mL of a mixture of 0.9% saline and 0.01%
merthiolate (SM). An aliquot of this mixture was added to a
pre-prepared IDENT stain tube before being assessed for
sperm count using CASA.
Groups 2 and 3 Samples frozen for possible future assessment
Homogenization-resistant
spermatid count-
Groups 1 and 4

After removal of the tunica, the left testis of each male was
homogenized for at least thirty seconds in 25 ml of SM. An
aliquot of this mixture was added to a pre-prepared IDENT
stain tube before being assessed for homogenization-
resistant spermatid count using CASA.
Groups 2 and 3 Samples frozen for possible future assessment.
3.7.4 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.
Full List All animals killed or dying prematurely.
All terminal animals of Groups 1 and 4 killed at a scheduled
interval.
Abnormalities only All terminal animals of Groups 2 and 3 killed at a scheduled
interval.
Routine staining Sections were stained with hematoxylin and eosin.
3.7.5 Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All animals from all groups. All specified in Section 3.7.
Scheduled kill All animals of Groups 1 and 4. All specified in Section 3.7.
All animals of Groups 2 and 3. Abnormalities 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.
3.7.6 Stage-dependent Evaluation of Spermatogenesis
Stage dependent evaluation of spermatogenesis was conducted on sections of testes from all
animals of Groups 1 (Control) and 4 (250 mg/kg/day) sacrificed on completion of the
scheduled treatment period prepared and stained using the PAS method. A qualitative
examination of spermatogenic stages was made for normal progression of the stages of the
spermatogenic cycle, cell associations, and proportions expected to be present during normal
spermatogenesis.


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3.8 Data Evaluation
This report contains serial observations pertaining to all weeks of study completed, together
with signs data collected during the necropsy period. In the case of clinical signs, body
weight and food consumption data, only information from the final week of the
acclimatization period is presented.
Summary statistics (e.g. means and standard deviations) presented in this report were
calculated from computer-stored individual raw data. Group mean values and standard
deviations were frequently calculated using a greater number of decimal places than
presented in the appendices. It is, therefore, not always possible to derive exact group values
from the data presented in the appendices.
Throughout the report the following abbreviations are used:
M Male
F Female
SD Standard deviation
N Number of animals/cages examined
Week of pretreatment relate to study weeks, as follows:
Phase week P1
Week of study -1
Days of pretreatment relate to study days, as follows:
Phase day P5 P6 P7 P8
Day of study -3 -2 -1 1*
* Prior to dosing

Statistics:

All statistical analyses were carried out separately for males and females using the individual
animal as the basic experimental unit.
The following data types were analyzed at each time point separately:
Grip strength and motor activity
Body weight, using gains over appropriate study periods
Hematology
Blood chemistry
Urinalysis
Sperm analysis
Organ weights, absolute or adjusted for terminal body weight
The following comparisons were performed:
Group 1 vs 2, 3 and 4
Group 1 vs 4 (where appropriate)
The following sequence of statistical tests was used for grip strength, motor activity, body
weight, sperm analysis, organ weight and clinical pathology data:
A parametric analysis was performed if Bartlett's test for variance homogeneity
(Bartlett, 1937) was not significant at the 1% level. 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 deviations of the observed means from the maximum
likelihood means, calculated under a constraint of monotonicity with the usual error
mean square, EMS. The null hypothesis is that the true means are monotonically
ordered. The test statistic is F1 = NMS/EMS which can be compared with standard
tables of the F distribution with 1 and error degrees of freedom. If the F1 approximate
test for monotonicity of dose-response was not significant at the 1% level, Williams'
test for a monotonic trend was applied. If the F1 approximate test was significant,
suggesting that the dose response was not monotone, Dunnett's test (Dunnett, 1955;
1964) was performed instead. Where there were only two groups, comparisons were
made using t-tests.
A non-parametric analysis was performed if Bartlett's test was still significant at the
1% level following both logarithmic and square-ro

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs of a reaction to treatment were restricted to chin rubbing and salivation in a few males
and females treated at 250 mg/kg/day and in isolated animals at 100 mg/kg/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test-article death. Female No. 101 administered 250 mg/kg/day was euthanized
for welfare reasons on Day 86 of treatment and had no significant macroscopic or
microscopic finding. Poor clinical condition was considered the major factor resulting in
early termination of this female.

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology examination of the peripheral blood during the 13th week of treatment revealed
higher than control mean prothrombin times in males at 10 mg/kg/day and in both males and
females at 100 or 250 mg/kg/day and lower than control mean activated partial
thromboplastin time in males at 10 or 100 mg/kg/day and in males and females at
250 mg/kg/day. Lower than control fibrinogen concentration was apparent in males and
females at 100 or 250 mg/kg/day.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Analysis of the urine during Week 13 revealed lower than control mean urinary creatinine
concentration in males and females receiving 250 mg/kg/day

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

250 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

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It was concluded that oral administration of Methanal, reaction products with 1,3-
Bis(aminomethyl)benzene and Hydroxybenzen to Sprague-Dawley rats over a period of
13 weeks at dose levels of 10, 100 or 250 mg/kg/day did not give rise a treatment related
effect.
Under the conditions of this study, the no observed adverse effect level (NOAEL) for
Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen
following oral gavage administration to Sprague-Dawley rats was considered to be
250 mg/kg/day.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen (an industrial chemical used in many chemical manufacturing processes) in a 13 week oral gavage study in Sprague- Dawley rats.
Three groups, each comprising ten male and ten female Sprague-Dawley rats, received Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen at doses of 10, 100 or 250 mg/kg/day. A similarly constituted control group received the vehicle, propylene glycol, at the same volume dose.
During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water consumption (by visual assessment), ophthalmic examination, hematology (peripheral blood), blood chemistry, urinalysis, thyroid hormone, estrous cycle, organ weight, sperm analysis, macropathology and histopathology investigations were undertaken.

Results
There was no test-article death and signs of a reaction to treatment were restricted to chin rubbing and salivation in animals at 100 or 250 mg/kg/day.
There was no effect of treatment on the condition, sensory reactivity, grip strength or motor activity. It was considered that bodyweight gain, food consumption and water intake were unaffected by treatment.
Ophthalmic examination during Week 13 indicated that there was no effect of treatment on the condition of the eyes.
Haematology examination of the peripheral blood during the 13th week of treatment revealed higher than control mean prothrombin times in males at 10 mg/kg/day and in both males and females at 100 or 250 mg/kg/day and lower than control mean activated partial
thromboplastin time in males at 10 or 100 mg/kg/day and in males and females at 250 mg/kg/day. Lower than control fibrinogen concentration was apparent in males and females at 100 or 250 mg/kg/day. There was no effect of treatment on the biochemical composition of the plasma during Week 13 of treatment and analysis of the urine revealed lower than control mean urinary creatinine concentration in males and females receiving 250 mg/kg/day
There was no effect of treatment on the stage of estrous of female animals or on the number, motility or morphology of the sperm from male animals treated at 250 mg/kg/day
After 13 weeks of treatment there was no effect of treatment on organ weights and nomacroscopic or histopathological abnormality.

Conclusion
Under the conditions of this study, the no observed adverse effect level (NOAEL) for Methanal, reaction products with 1,3-Bis(aminomethyl)benzene and Hydroxybenzen following oral gavage administration to Sprague-Dawley rats was considered to be 250 mg/kg/day.