6-phenyl-1,3,5-triazine-2,4-diyldiamine

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

systemic toxicity of parental animals occurs along with effects on offspring.

clearly reprotoxic effects include decreased fertility and gestation indices, reduced lack of nursing activity and agalactia.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

The study was initiated as DRF for an EOGRTS.

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

October 22, 2021- November 18, 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

additionally 70 days premating dosing, no clinical chemistry and hematological profile performed

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The test design was chosen as the study was intented to seve as a DRF study for an Extended One Generation Reproductive Toxicity Study.

Specific details on test material used for the study:

Lot No 023302
Manufacturing date: November 30, 2020
Re-rest date: November 30, 2022

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is
designed to use the rat. The Wistar rat was selected due to large experience with this strain
of rat in reproduction toxicity studies and known fertility and availability of historical
control data at Test Facility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90. Hungary
Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good
conventional environment during the study.
Age of animals at start of Male animals: 46 – 51 days
the study: Female animals: 39 – 45 days
Body weights at start of 212 – 240 g for male animals
the study: 127 – 158 g for female animals
The weight variation did not exceed ± 20 per cent of the mean
weight
Number of animals
involved in the study: 48 males, 48 females (nulliparous, non-pregnant females)
Number of animals/group 12 animals/sex in the control and dose groups (at least
8 pregnant female animals per group were expected)
Number of groups: 4 (3 dose levels + 1 control group)
Acclimatization time: 6 days

Animal health: Only healthy animals were used for the study. Healthy status
was certified by the breeder (Appendix 18) and was controlled
before the start of the treatment.
Animal room no.: 24
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female / cage
Mated females: individually
Males after mating: 2 animals / cage
Cage type: Type III polypropylene/polycarbonate
Bedding: Certified laboratory wood bedding (SAFE 3/4-S-FASERN or
SAFE 3/4 S) produced by J. Rettenmaier & Söhne
GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany).
The bedding is suitable as nesting material. Details of quality of
bedding material were reported (see Appendix 21).
The cages and bedding were changed once or twice a week.
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Environmental conditions were maintained by an air-conditioning system. Temperature and
relative humidity were verified and recorded daily during the study.

Route of administration:

oral: gavage

Vehicle:

other: sunflower oil (Helianthi annui oleum raffinatum)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine) was formulated in the vehicle
(sunflower oil) in concentrations of 2.5, 25 and 62.5 mg/mL. Formulations were prepared in
the formulation laboratory of Test Facility on the basis of the stability features of the test
item in the vehicle for not longer than for three days and formulations were stored at room
temperature until use.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Due to the low solubility of the test item in water, sunflower oil was used as the vehicle. The
test item is suitable for oral administration when formulated in sunflower oil.
-Name: Sunflower oil (Helianthi annui oleum raffinatum)
Batch number 1: 8006975004
Expiry date 1: March 31, 2022
Batch number 2: 8007816003
Expiry date 2: August 31, 2022
Batch number 3: 8007907002
Expiry date 3: October 31, 2022
Supplier: Magilab Kft.
Király utca 12.
Budapest
H-1061 Hungary
Storage conditions: Below 25 °C, protected from light

Details on mating procedure:

Mating begun 10 weeks after the initiation of treatment with one female and one male of the
same dose group (1:1 mating) placed in a single cage. Females remained with the same male
until copulation occurred or 14 days had elapsed.
Male animal no. 403 was early euthanized therefore it was replaced by a proven male from
the same group (125 mg/kg bw/day). Mating partners were changed for one female at
50 mg/kg bw/day (no. 330) and for 5 female animals at 125 mg/kg bw/day (no. 422, 424,
425, 427 and 431) within their groups because most of these female animals showed regular
cycle but copulation did not occur during 14 days. Therefore, mating period was prolonged
to achieve the appropriate number of pregnant at the high dose group.
Vaginal smears were examined for the presence of vaginal plug or sperm. Presence of
vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0
of pregnancy as defined by OECD 421). Sperm positive females were caged individually.
Mating pairs were clearly identified in the raw data.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (verification of concentrations and homogeneity)
was performed in the Analytical Laboratory of Test Facility four times during the study.
Five aliquots of 10 mL of each formulation and five aliquots of 10 mL control substance
(vehicle) were taken and analyzed.
Date of sampling: December 06, 2021, January 05, 2022, January 31, 2022, February 08, 2022
Date of analysis: December 07, 2021, January 07, 2022, February 02, 2022, February 10, 2022
Concentration of the test item in the dosing formulations varied in the range of 91.6 and
99.5 % of the nominal values at each analytical occasion. Results and details of analysis are
attached to this Report (Appendix 17.2).
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended
concentrations was analytically verified by a validated HPLC method with UV detection up front.
The recovery of the test item from the vehicle was within the acceptance criteria – 98.3, 92.1
and 90.3 % relative to nominal concentrations at 1 mg/mL, 10 mg/mL and 200 mg/mL,
respectively.
6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine) proved to be stable in sunflower oil
at the intended concentrations at room temperature for three days.
Separate analytical reports provided details of analysis (Study no. 849-100-6171 and 849-
100-6432; GLP).

Duration of treatment / exposure:

The experimental period involved 6 days of acclimatization, 70 days pre-mating period,
mating and optional post-mating period for male animals, 70 days pre-mating period,
mating, gestation and lactation periods for female animals and necropsy day followed by
additional processing.
The end of the experimental period is the last day when raw data is generated after the
in-life period.
The day of first treatment was considered as day 0 of examination.

Frequency of treatment:

daily

Details on study schedule:

Males: Acclimatization 6 days
Pre-mating period 70 days
Mating period 28 days
Postmating period 6-33 days

Females: Acclimatization 6 days
Pre-mating period 70 days
Mating period 28 days
Gestation period 22-24 days
Lactation period 21 days

Dose / conc.:

125 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: according to available information from repeated dose toxicity studies
- Rationale for animal assignment (if not random): randomisation
- Fasting period before blood sampling for clinical biochemistry: not stated

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

Oestrous cyclicity (parental animals):

yes

Litter observations:

Observation of the delivery process



Females were allowed to litter and rear their offspring. Delivery process was observed as
carefully as possible from gestational day 21 onwards. All observations and any evidence of
abnormal deliveries were considered. The duration of gestation was recorded and was
calculated from day 0 of pregnancy.
Dams were observed whether they made a nest from the bedding material and nurse their
new-born or not. The sucking success was observed by the presence of milk in the pups'
stomach. All observations were recorded individually for each dam.
On post-partum day 4, the size of each litter was adjusted to four pups per sex per litter, if it
was feasible. Extra pups were eliminated by a random selection. Partial adjustment of litter
size was performed if the number of male and female pups did not allow having four of each
sex per litter (for example, five males and three females).
No pups were eliminated when litter size would have dropped below 10 pups/litter.



Each litter was examined as soon as possible after delivery (within 24 h of parturition), to
establish the number and gender of pups, stillbirths, live births, runts (pups that are
significantly smaller than normal pups) and the presence of gross abnormalities.
Live pups were counted, sexed, and litters weighed within 24 hours of parturition (on the
day after parturition is complete, day 0), and on days 7, 14 and 21 post-partum with an
accuracy of 0.1 g. Any abnormal behavior of the offspring was recorded.
On the day of birth, pups found dead were subjected to a lung flotation test to differentiate
pups that died in utero (stillborn; negative lung flotation test) from pups that died after birth
(dead pups; positive lung flotation test).
All litters were checked and observed, the number of viable and dead pups were recorded
daily. Dead pups found were subjected to necropsy by macroscopic examination. All
observed abnormalities were recorded.
The anogenital distance of each pup were determined on post-natal day 4. The anogenital
distance were normalized to the cube root of the body weight. Therefore, individual body
weight of pups was determined with an accuracy of 0.01 g on post-natal day 4. (The litter
weight was calculated for evaluation on post-natal day 4).
The number of nipples/areolae in male pups were counted on post-natal day 13.

Postmortem examinations (parental animals):

Necropsy
Gross necropsy was performed on each animal immediately after death or one day after the
last treatment. Animals were euthanized by exsanguination after verification of deep narcosis by Isofluran
CP® (details are presented in paragraph “Characteristics of anesthetics”) and subjected to
gross necropsy as follows:
- Dead female animal at 5 mg/kg bw/day: Day 111 (lactation day 14)
- Male animal euthanized because of its poor condition at 125 mg/kg bw/day: Day 72
- Parental male animals: on Day 104
- Dams: on lactation days 22-24, Days 114-120
- Dams for which no living pups remained: Days 97-103 (lactation days 1-5)
- Not delivered females: on Days 107, 111 (gestation days 23 and 24)
- Non-pregnant female: on Day 111, 24 days after mating
- Not mated females: Day 97
- Offspring: on post-natal days 22-24, offspring of dead dam on post-natal day 14
After examination of the external appearance, the cranial, thoracic and abdominal cavities
were opened and the appearance of all tissues and organs was observed, and any
abnormality was recorded including details of the location, color, shape and size. Special
attention was paid to the organs of the reproductive system.
The number of implantation sites was recorded.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and
seminal vesicles with coagulating glands and stomach of all adult animals were preserved.
Testes and epididymides were preserved in modified Davidson solution, all other organs in
4 % buffered formaldehyde solution.
Thyroid gland was preserved from all adult males and females and from one male and one
female pup per litter for the intended subsequent histopathological examination. Thyroid and
parathyroid were preserved together with pharynx.
All organs showing macroscopic lesions were also preserved in 4 % buffered formaldehyde
solution.
Based on macroscopic observations, the spleen, liver, kidneys, thymus and stomach, liver
and stomach were preserved in all male animals in each group and the liver of all female
animals was also preserved in all groups.

Organ weight
At the time of termination, body weight, brain weight, weight of the testes, epididymides
and prostate as well as seminal vesicles with coagulating glands as a whole of adult male
animals were determined. Paired organs were weighed together. Absolute organ weight was
recorded. Relative (to body and brain weight) organ weights were calculated and reported.
The thyroid weight was not determined.
Thymus and spleen, which were judged to be smaller than normal were also weighed for a
possible evaluation (not reported yet)

Histopathology



Detailed histological examinations were performed on the ovaries, uterus with vagina, testes,
epididymides – with special emphasis on stages of spermatogenesis in the male gonads and
histopathology of interstitial testicular cell structure – in the animals in the control and high dose
groups. Detailed histological examination of the ovaries covered the follicular, luteal, and
interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histological processing and evaluation of organs and tissues were performed in dead female
animal (1/12 at 5 mg/kg bw/day) and in male animal euthanized early (1/12 at 125 mg/kg
bw/day). The thyroid glands were also processed and evaluated histologically in all male
and female animals in each group based on changes of thyroid hormone levels.
In addition, the following organs were processed and examined histologically based on the
macroscopic findings:
- Control group: kidneys - 2/12 male, skin - 2/12 male,
- 5 mg/kg bw/day: kidneys - 1/12 male,
- 50 mg/kg bw/day: epididymides, testes - 1/12 male;
- kidneys - 4/12 male, 1/1 not mated female
- mammary gland – 1/11 dam
- ovaries, uterus, vagina – 1/11 dam, 1/1 not mated female
- 125 mg/kg bw/day:
- kidneys – 4/11 male, 1/7 dam
- liver – 4/7, dam, 2/2 not delivered female, 2/2 not mated female, 1/1 non-pregnant
female
- mammary gland – 2/2 not delivered pregnant female
- spleen – 6/11 male
- thymus – 8/11 male, 3/7 dam, 1/1 non-pregnant female
The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a
microtome (at a thickness of 2-4 µm), placed on glass microscope slides, stained with
hematoxylin and eosin and examined by light microscopy.

Postmortem examinations (offspring):

Pups selected for thyroid gland preservation (one male and one female pup per litter, where
it was feasible) were subjected to gross macroscopic observations.
Pups euthanized on post-natal day 4 or 13 were carefully examined externally for gross
abnormalities.
Dead pups were subjected to necropsy immediately after they were found dead.
Selected organs and tissues were excised, trimmed of any adherent tissue, as appropriate,
weighed and preserved as described above.

Statistics:

The statistical evaluation of appropriate data (marked †above) was performed with the
statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of
variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA)
is carried out.
If the obtained result was significant Duncan Multiple Range test was used to access the
significance of inter-group differences. Getting significant result at Bartlett’s test, the
Kruskal-Wallis analysis of variance was used and the inter-group comparisons were
performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose
was calculated.

Reproductive indices:

All parameters required in the OECD 421 guideline were evaluated and reported
respectively. The following parameters were examined and evaluated:
Parental males



− Clinical observations
− Body weight †
− Body weight gain †
− Food consumption †
− Percentage of pairings
− Percentage of fertile males †
− Percentage of infertile males
− * Male copulatory index †
− * Male fertility index †
− Thyroid hormone (FT3, FT4 and TSH) level†
− Necropsy findings
− Organ weights (absolute and relative to the body and brain weights) †
− Histopathology findings



Parental females



− Clinical observations
− Body weight †
− Body weight gain †
− Food consumption †
− Percentage of pairings /sperm positive females
− Percentage of pregnant females
− Percentage of sperm positive, but non-pregnant females
− Percentage of non-mated females
− * Female copulatory index †
− * Female fertility index †
− * Gestation index †
− Duration of pregnancy (days) †
− Number of implantations / dams †
− * Post-implantation mortality †
− Number of dams with live pups on lactation days 0, 4, 7, 14 and 21
− FT3, FT4 and TSH levels on post-partum day 13
− Necropsy findings
− Histopathology findings

Offspring viability indices:

All parameters required in the OECD 421 guideline were evaluated and reported
respectively. The following parameters were examined and evaluated:
Offspring



− Litter weight and body weight on post-natal days 0, 4, 7, 14 and 21 †
− Litter weight gain and mean body weight gain per litter between post-natal days 0-7, 7-
14 and 14-21 and for overall post-natal days †
− Number of live births per litter, and number of viable pups per litter on post-natal days 0,
4 and 21 †
− * Survival Index of pups on post-natal day 4 †
− * Sex ratio % (on post-natal days 0 and 4) †
− Normalized anogenital distance †
− Number of nipples/areolae in male pups †
− Thyroid hormone (FT3, FT4 and TSH) level of pups on post-natal days 4 and 13 †



* = Formulas for calculations are given below
† = Statistical analysis was performed

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related clinical signs (decreased activity, piloerection) were detected in most of the
male animals at 125 mg/kg bw/day by the end of the treatment period and in the female
animals at 125 mg/kg bw/day with lower incidence at the end of pre-mating period and
during gestation period. Emaciation was also noted for some male and female animals at the
daily clinical observations at 125 mg/kg bw/day.
The behavior and physical condition were predominantly normal in the control, 5 and
50 mg/kg bw/day groups in male animals during the pre-mating, mating and post-mating
periods and in female animals during the pre-mating, mating, gestation and lactation periods.

The number of dams with adequate nursing instinct was clearly and dose-dependently
reduced at 50 and 125 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality



One female animal died during the course of the study (no. 226, 1/12 at 5 mg/kg bw/day) on
lactation day 14. There were no preceding clinical signs or body weight change.
Based on histopathology findings, the cause of the death was shock and suffocation
presumably due to mis-gavage at the test item administration.
One male animal at 125 mg/kg bw/day (no, 405, 1/12) was early euthanized because of its
poor condition on Day 72. Significant body weight loss, piloerection and decreased activity
were detected during the preceding days. Based on necropsy and histopathological findings,
enteral lesions caused poor state of this animal.
There was no mortality in the control, 5 or 50 mg/kg bw/day groups during the entire
observation period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was dose dependently depressed in male and female animals at
50 and 125 mg/kg bw/day during the entire treatment period.
The mean body weight and body weight gain were mostly comparable to the control at
5 mg/kg bw/day in male animals from Day 0 up to and including Day 103 as well as in female
animals during the premating, gestation and lactation periods. Statistical significance with
respect to the control was detected in female animals at 5 mg/kg bw/day in terms of lower
mean body weight gain between Days 35 and 42. This minor change in the body weight gain
did not result in changes in the mean body weight of low dose treated female animals,
therefore, this finding was considered to have no toxicological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean daily food consumption was reduced in male animals at 125 mg/kg bw/day.
A slight reduction of the mean daily food consumption was detected in male animals at
5 mg/kg bw/day on week 4 and at 50 mg/kg bw/day on weeks 1, 4, 8 and 15.
In the male animals at 125 mg/kg bw/day, the mean daily food intake was consistently lower
than in the control group mostly during the entire observation period reaching statistical
significances on weeks 1, 3, 5-10, 14 and 15.
The mean daily food consumption of female animals was similar in the control, 5, 50 and
125 mg/kg bw/day groups during the entire pre-mating period except for 125 mg/kg bw/day
on week 1.
Statistical significance with respect to the control was observed at the slightly lower mean
daily food consumption in female animals at 50 mg/kg bw/day on lactation week 1 and at
125 mg/kg bw/day between gestation days 7-14 and 14-21.
This minor although statistically significant change in the mean daily food consumption was
judged to be toxicologically not relevant in male animals at 5 or 50 mg/kg bw/day and in the
female animals in each dosed group due to the low degree and transient occurrence.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

In principle, the thyroid hormone (FT3, FT4 and TSH) levels were considered to be not
directly adversely affected in parental animals or in offspring at any dose levels.
Nevertheless, the test item exposure led to thyroid hormone changes in parental animals,
which were more pronounced in males than in females. These were noted at 50 mg/kg
bw/day (male) and 125 mg/kg bw/day (male and female) groups. However, these changes
have to be evaluated in respect to the observed general impairment of the health status.
Effects on body weight/body weight gain, selected clinical as well as macroscopic and
histopathological findings indicated a poor general health at these dose levels. When
changed, the thyroid hormones (FT3, FT4 and TSH) were predominantly consistently
reduced instead of down (FT3, FT4) and up (TSH) regulated in parallel or vice versa.
Moreover, there were no test item-related changes in the weights of thyroid glands
(measured male animals) and cell morphology of thyroid glands in male and female animals
at 5, 50 or 125 mg/kg bw/day. Therefore, the observed variations in the serum thyroid
hormone levels were finally considered to be secondary in nature.
More precisely, in the parental male animals, lowered mean concentrations were detected for
FT3 at 125 mg/kg bw/day and for FT4 and TSH at 50 and 125 mg/kg bw/day when compared to
the control.
In the parental female animals, the mean FT3 concentration exceeded the control at
50 mg/kg bw/day while the mean concentrations of FT4 and TSH (no statistical
significance) were below the control at 125 mg/kg bw/day.
No statistically significant no biologically relevant differences with respect to the control
were observed in the thyroid hormone concentrations in PND4 (FT3 and FT4) or PND13
(FT3, FT4 or TSH) offspring at 5 or 50 mg/kg bw/day.
Determination of thyroid hormones was not feasible in pups at 125 mg/kg bw/day as no
living pups remained from post-natal day 4.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological investigation did not reveal test item related adverse lesions detectable by
microscopic examination of the investigated reproductive organs of all exposed
experimental male and female animals at 125 mg/kg bw/day.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No morphological evidence of test item-related injury (degeneration, inflammation, necrosis
etc.) of the liver, the pancreas, the cardiovascular system, the respiratory system, the urinary
system, the immune system, the hematopoietic system, the skeleton, the muscular system,
the central, or peripheral nervous system, the eyes, the integumentary system, the
reproductive system was observed at any dose level.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significant differences with respect to the control were observed at 50 mg/kg
bw/day in the form of a lower number of animals with regular cycles. At this dose also a
lower mean number of cycles, longer mean length of cycles, lower mean number of days in
pro-estrous and estrous as well as higher mean number of days in diestrus were noted
attaining statistical significance. At125 mg/kg bw/day the statistically significant deviations
were recorded for a lower mean number of cycles and lower mean number of days in estrous
and diestrus.
Although the data are within or close to the ranges of historical control values, the changes
were not related to doses and there was a lack of related findings in the reproductive
parameters, a test item exposure related effect cannot be excluded with certainty.
Test item exposure related influence on the estrous cycle was not detected at 5 mg/kg
bw/day by.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The reproductive performance (copulatory, fertility and gestation indices) was clearly
impaired in male and female animals at 125 mg/kg bw/day and females at 50 mg/kg bw/day
(copulatory index).
The reproductive performance at 5 mg/kg bw/day was comparable to the control.
At 125 mg/kg bw/day, the copulatory and fertility indices (percentage of mated animals and
percentage of fertile pairing or pregnant female animals) were significantly lower than in the
control group in male or female animals. Percentage of delivered pregnant animals (gestation
index) was also significantly lower than in the control group and historical control.
Statistical significance was also detected for the lower copulatory index at 50 mg/kg bw/day
(male and female) when compared to the control. Although the copulatory index was at the
lower end of the historical control ranges, it cannot be excluded with certainty whether this
dose-dependent observation has a toxicological relevance. Therefore, a test item relationship
was finally considered as likely. Fertility and gestation indices were not affected.

The delivery data were impaired at 125 mg/kg bw/day (mean number of total births and
alive pups, as well as higher mean number of prenatal loss).
There were no significant differences between the control and 5 or 50 mg/kg bw/day
administered groups in the mean number of implantations, pre- and post-natal loss or in total
loss, number of pregnant animals, dams delivered, mean duration of pregnancy, mean number
of births (total, live and stillborn) or in the live birth indices.
In the female animals at 125 mg/kg bw/day, the mean number of total birth and alive pups
were significantly lower and the mean number of pre-natal loss (total loss) was significantly
higher than in the control group.
The number of pregnant animals and dams that delivered were also slightly lower comparing
to actual control or historical control, while the mean duration of pregnancy and mean number
of implantation sites were similar to the control in female animals at 125 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

gross pathology

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs were detected with high incidence in the offspring at 50 and 125 mg/kg
bw/day. Cold, not suckled, dead and missing offspring were detected at the clinical
observations in mid and high dose groups during the first post-natal day due to inadequate
maternal nursing.
The percentage of offspring with clinical signs was not related to dosing at 5 mg/kg bw/day
group.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

No living pups remained at 125 mg/kg bw/day therefore, evaluation was only conducted for
5 and 50 mg/kg bw/day groups comparing to control from post-natal day 4.
The extrauterine mortality was markedly higher at 50 and 125 mg/kg bw/day due to the poor
general conditions of mothers (lack of milk in mammary gland, inadequate nursing).
There was no test item-related effect on offspring’s extra uterine mortality at 5 mg/kg bw/day.
The mean number of live pups per litter and the mean number of viable pups per litter were
similar in control and 5 mg/kg bw/day groups on post-natal days 0, 4 and 21. The survival
indices were also comparable to control at 5 mg/kg bw/day.
The mortality rate was extremely high at 50 mg/kg bw/day (44 %) and at 125 mg/kg bw/day
(100 %) on post-natal day 4, thus the survival index was significantly lowered in 50 mg/kg
bw/day group on post-natal day 21.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight of the offspring was significantly reduced at 50 and 125 mg/kg bw/day at
birth. The body weight development was also lowered at 50 mg/kg bw/day during the
observation period from birth to post-natal day 21.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distances (absolute and normalized) were not adversely affected in male or
female offspring at 5 or 50 mg/kg bw/day.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

There was no nipple in male offspring in control, 5 or 50 mg/kg bw/day on PND13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Specific macroscopic alterations were not found in offspring subjected to gross pathological
examinations (stillborn or dead pups before PND 22 or pups at termination).
There were no macroscopic alterations in stillborn pups on post-natal day 0. One pup at
125 mg/kg bw/day was partially cannibalized (only head remained).
In dead pups, empty stomach (1/1 control, 6/13 at 50 mg/kg bw/day, 23/28 at 125 mg/kg
bw/day), autolyzed visceral organs (8/13 at 50 mg/kg bw/day, 6/28 at 125 mg/kg bw/day),
cannibalized organism and paleness (1/28 at 125 mg/kg bw/day) were detected.
Terminally (on post-natal days 14-24), the organs and tissues were normal in pups subjected to
necropsy observations: 23/23 control, 24/24 at 5 mg/kg bw/day, 16/16 at 50 mg/kg bw/day.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex distribution
The ratio and litter mean of genders were similar in the control and test item-administered
groups on post-natal day 0, and at 5 and 50 mg/kg bw/day on PND 4 and PND 21.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other: Effects on offspring are strongly related to lack of nursing activity and effects on mammary glands of parent animals

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

50 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

presumably yes

Conclusions:

Under the conditions of the present Reproduction/Developmental Toxicity Screening
Test, exposure to 6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine) caused
systemic toxicity in the form of clinical signs, depressed body weight development
(male and female), reduced food consumption (male), macroscopic changes
(gastrointestinal tract, lymphoid organs, mammary gland, male or female), changes in
organ weights (male), histological findings (gastrointestinal tract, lymphoid atrophy,
hypo-lactation) in Han:WIST rats at 125 mg/kg bw/day by oral gavage as investigated
in this study.
At 50 mg/kg bw/day, depressed body weight development (more pronounced in males
and less consistent in females), macroscopic and histopathology changes in mammary
gland (female) and changes in organ weights (male) were observed.
There were no test item-related effects at 5 mg/kg bw/day (male and female).

6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine) impaired the reproductive
performance (prolongation of mating period, copulatory, fertility and gestation
indices, nursing instinct) in male or female animals at 125 mg/kg bw/day. In females,
also differences in the estrous cycle were recorded.
At 50 mg/kg bw/day, an isolated case of mating period prolongation occurred and the
nursing instinct of several dams decreased mostly on lactation days 0 and 1 as well as a
reduction of the copulatory index in both sexes was noted. However, the reproductive
performance (gonad function, mating behavior, conception, parturition) was normal in
parental male and female Han:WIST rats at 50 mg/kg bw/day except for an indication
of estrous cycle impairment in females as far as investigated in this study.
The reproductive performance was normal, i.e., characteristic on the strain of this
species at 5 mg/kg bw/day (male and female).
The development of the F1 offspring (mortality, clinical signs, body weight) was
adversely affected from birth up to post-natal day 21 after repeated oral
administration of dams at 50 and 125 mg/kg bw/day. Moreover, significantly higher
pre-natal loss causing a reduced number of viable pups was observed at 125 mg/kg
bw/day, which is considered to be an effect on the development of F1 offspring.

Based on these observations, the No Observed Adverse Effect Levels (NOAEL) were
determined as follows:
NOAEL for systemic toxicity of male/female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of male/female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of female rats: 5 mg/kg bw/day
NOAEL for F1 offspring development: 5 mg/kg bw/day

Executive summary:

The objective of this study was to obtain initial information on the possible effects of the
test item on reproduction and development when repeatedly administered orally (by gavage)
to rats at doses of 5, 50 and 125 mg/kg bw/day compared to control animals according to
OECD 421. The guideline is designed for use with the rat, which is the preferred rodent
species for toxicity and reproduction toxicity testing.
As a screening test, it intended to provide initial information on possible effects on male and
female reproductive performance such as gonadal function, mating behavior, conception,
pregnancy, parturition as well as on development of the F1 offspring from conception to
post-natal day 21 associated with administration of repeated maternal doses and to allow a
dose-setting for a possible subsequent Extended One-Generation Reproduction Toxicity
Study (main study according to OECD 443).
Four groups of Han:WIST rats consisting of 12 animals per group and sex were administered
orally (by gavage) once daily at 0 (vehicle only), 5, 50 and 125 mg/kg bw/day doses in
concentrations of 2.5, 25 and 62.5 mg/mL corresponding to a 2 mL/kg bw dosing volume. A
group of vehicle (sunflower oil) treated animals (n= 12/sex) served as a control.
The suitability of the vehicle for the test item was analytically verified up front.
The concentration of the test item in the dosing formulations administered to animals was
checked four times during the study. 6-phenyl-1,3,5-triazine-2,4-diamine (Benzoguanamine)
concentrations in the dosing formulations varied in the acceptable range between 91.6 % and
99.5 % of the nominal values and confirming the proper preparation of the dosing
formulations.
All animals of the parent (P) generation were dosed prior to mating (70 days) and
throughout mating. In addition, males received the test item or vehicle after mating up to the
day before the necropsy (altogether for 104 days). Female animals were additionally
exposed through the gestation and lactation periods, i.e., up to the day before necropsy
(altogether for 97-120 days, depending on the effectiveness of pairing).
Observations included mortality, clinical signs, body weight, food consumption, mating,
pregnancy and delivery process, as well as development of offspring. Estrous cycle was
monitored by examining vaginal smears before the mating for two weeks and during the
mating period until evidence of mating, as well as on the day of the necropsy. Blood samples
were collected for determination of serum levels of thyroid hormones (FT3, FT4 and TSH)
from 2-6 pups per litter (where it was feasible) on post-natal day 4, from all dams and
2-4 pups per litter on post-partum/ post-natal day 13, from not mated, non-pregnant and not
delivered female animals on day of necropsy, from all parental male animals on Day 104.
The dams were allowed to litter and rear their offspring at least up to day 21 post-partum.
Litters were weighed and offspring were observed for possible abnormalities and were
euthanized on post-natal day 22 or shortly thereafter.

All parental animals were subjected to gross pathology one day after the last treatment. The
sexual organs (ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda),
prostate and seminal vesicles with coagulating glands) and all organs showing macroscopic
lesions of all adult animals were preserved. Based on the necropsy observations, the liver,
spleen, kidneys, stomach and thymus of all male animals and liver of all female animals in
each group were also fixed in 4 % buffered formaldehyde solution for further possible
histological examinations.
Thyroid gland was preserved from all adult males and females and from one male and one
female pup per litter for the possible subsequent histopathological examination. Thyroid
glands were weighed in all male animals and processed histologically in all male and female
animals based on observed changes in thyroid hormone levels.
Gross necropsy was performed for offspring selected for thyroid gland preservation.
The body weight, brain weight and weight of the testes, epididymides and prostate and
seminal vesicles with coagulating glands as a whole of adult male animals were determined.
Histopathology examination was performed on ovaries, testes, epididymides in control and
high dose groups. Additionally, full histopathological examinations were performed in organs
and tissues of dead and early euthanized animals as well as in organs with macroscopic
findings were processed and evaluated histologically (kidneys, mammary gland, liver, skin,
spleen or thymus).
The results of this study were summarized as follows:
Mortality
One male animal at 125 mg/kg bw/day was early euthanized because of its poor general
condition on Day 72. Based on necropsy and histopathological observations, test item
induced enteral lesions caused the poor state of this male animal.
One female animal at 5 mg/kg bw/day died on lactation day 14 presumably due to mis-
gavage at the test item administration. Pulmonary lesions (severe congestion and alveolar
emphysema) refer to shock and suffocation leading to death in this female animal.
Clinical observation
Test item related clinical signs (decreased activity, piloerection) were detected in most of the
male animals at 125 mg/kg bw/day by the end of the treatment period and in the female
animals at 125 mg/kg bw/day with lower incidence at the end of pre-mating period and
during gestation period. Emaciation was also noted for some male and female animals at the
daily clinical observations at 125 mg/kg bw/day.
Body weight and body weight gain
The body weight development was dose dependently depressed in male and female animals
at 50 and 125 mg/kg bw/day during the entire treatment period.
Food consumption
The mean daily food consumption was reduced in male animals at 125 mg/kg bw/day.
The slight reduction of the mean daily food consumption was transient and was in full
accordance with body weight changes in male animals at 50 mg/kg bw/day and in female
animals at 50 and 125 mg/kg bw/day. Therefore, this minor change was judged to have little
or no toxicological relevance.

Estrous cycle
Statistically significant differences were observed at 50 mg/kg bw/day with regards to a
lower number of animals with regular cycles, lower mean number of cycles, longer mean
length of cycles, lower mean number of days in pro-estrous and estrous as well as higher
mean number of days in diestrus. At 125 mg/kg bw/day statistically significant deviations
were recorded for a lower mean number of cycles and lower mean number of days in estrous
and diestrus.
Pregnancy and delivery data of dams
The delivery data (mean number of total births and alive pups, as well as mean number of
prenatal loss) were impaired at 125 mg/kg bw/day. The number of births and alive pups was
below the control along with significantly higher prenatal loss.
The nursing instinct of dams ceased at 50 and 125 mg/kg bw/day dose dependently due to
the worse general condition and lack of milk in the mammary gland.
Reproductive performance
The reproductive performance was clearly impaired in male and female animals at
125 mg/kg bw/day and to a lesser extent in females at 50 mg/kg bw/day (copulatory index).
The copulatory and fertility indices (percentage of mated animals and percentage of fertile
pairing or pregnant female animals) and gestation index (percentage of pregnancy that
provides at least one live pup) was severely reduced.
The mating period was markedly elongated at 125 mg/kg bw/day and in a single case at 50
mg/kg bw/day.
Serum thyroid hormones
The thyroid hormone (FT3, FT4 and TSH) levels were considered to be not adversely
affected in parental animals or in offspring at any dose levels.
Nevertheless, the test item exposure led to thyroid hormone changes in parental animals,
which were more pronounced in males than in females. These were finally supposed to be
related to the various findings indicative for a poor general health status. Moreover, there
were no test item-related changes in the weights of thyroid glands (measured in male
animals) and cell morphology of thyroid glands in male and female animals at 5, 50 or
125 mg/kg bw/day.
Necropsy
Gross necropsy revealed test item-related macroscopic findings in the gastrointestinal tract
(stomach, intestines, mesenteric lymph nodes), in lymphoid organs (spleen, thymus) in male
animals at 125 mg/kg bw/day. Dilatation of stomach, undigested food in the stomach,
thickened intestines or enlarged mesenteric lymph nodes were detected in these animals at
the necropsy observations,
There was no milk in the mammary glands with a dose related incidence in several dams at
50 and 125 mg/kg bw/day and some female animals were emaciated at 125 mg/kg bw/day.
Organ weight
Evaluation of organ weights revealed dose related significant changes in the examined
organs – testes, epididymides, seminal vesicle with coagulating gland or prostate as a whole
(absolute, relative to body or brain weight) – at 50 and 125 mg/kg bw/day.

The differences with respect to the control were considered to reflect mainly consequences
of the markedly lowered mean fasted body weight of male animals in mid and high dose
groups as there were no related morphological changes in these organs at the histopathology.
The mean weights of thyroid glands were not affected in the investigated male animals at
any dose level.
Histopathology
The histological structure of the investigated reproductive organs – testes, epididymides
ovaries and uterus – was characteristic for a normal functional activity in all exposed
parental male and female animals in the control and 125 mg/kg bw/day group.
125 mg/kg bw/day dose caused gastro-intestinal findings in a proportion of experimental
animals, lymphoid atrophy in the spleen and thymus and hypolactation in the mammary
gland in accordance with macroscopic observations.
The cell morphology of thyroid gland was not affected in all male and female animals in the
control, 5, 50 and 125 mg/kg bw/day groups at the histopathological investigations.
Offspring
The offspring’s development was significantly impaired at 50 and 125 mg/kg bw/day. The
high extrauterine mortality, high incidence of clinical signs (cold body temperature, not
suckled, found dead, missing) and significantly reduced body weight development were
presumably due to the worse general condition of dams and lack of maternal nursing instinct
at birth or shortly thereafter at 50 and 125 mg/kg bw/day.
No adverse effects on mortality, clinical signs, body weight, anogenital distance, nipple
retention or necropsy were detected in the offspring at 5 mg/kg bw/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

CMC (carboxymethyl cellulose)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

male; 49 days: female; for 14 days before mating to day 3 of lactation

Frequency of treatment:

one administration/day

Details on study schedule:

Premating exposure period :
Male : 14 day
Female : 14 day
Duration of test: male: 49 day
female: 39-53 day

Remarks:

Doses / Concentrations:
0, 4, 20, 100 mg/kg bw/day
Basis:

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no effects observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

other: lack of nursing activity

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

not specified

Basis for effect level:

viability

body weight and weight gain

Reproductive effects observed:

not specified

The NOAEL of 4 mg/kg bw/day for breast feeding females cannot be assigned to a reproductive toxicity unambigeously.

The lack of nursing activity can be due to the general stress caused by the toxicity of the substance or to specific effects of Benzoguanamine on the lactation.

Treatment related changes were only revealed after delivery of F₀dams due to poor collection, nursing and heating for newborns at 20 mg/kg/day and more group.

STATISTICAL RESULTS: As for reproductive performance, no effects related to this substance were observed for the estrous cycle, numbers of corpora lutea and implantation, copulation index, conception index, and duration of mating. On examination after delivery, poor collection and heating of new borns were observed with dams of the 100 mg/kg group. Furthermore, the birth index decreased with increase of stillborns at this dose. No effects related to this substance were observed in terms of gestational days, number of litters and live newborns, gestation index and sex ratio. There were no external anomalies of pups. Examination during the lactation period, revealed poor collection, nursing and heating for newborns. A decrease in the viability index on day 4 of lactation was observed for dams of the 20 mg/kg or more group.

Findings of delivery of Fo dams treated orally with this subsutance

Dose (mg/kg)		0			4	20		100
No. of copulated (male, female) No. of impregnated (female)		12 11			12 12	12 12		12 11
No. of dams		11			12	12		10
Gestation index		100.00			100.00	100.00		90.90
No. of poor nursing dams
on day 0		0			0	0		2^1)
day 4		0			0	2^2)		7^2)
Mean no. ofnewborns/litter		15.45			14.45	13.75		15.20
S.D. of number		3.14			1.66	3.70		2.25
No. of stillborns		0			4*	4		43**
No. of live newborns		170		173			161		109
Mean no. of live newborns/litter		15.45			14.42	13.43		10.90
S.D. of number		3.14			1.62	3.45		5.07
Birth index		100.00			97.74	97.58		71.71**
Sex ratio of live newborns		0.87			0.90	0.96		0.98
Body weight of live newborns (g)
male on day 0		6.3			6.8	6.2		5.1**
day 4		9.6			10.6	9.9		6.8**
female on day 0		6.0			6.5	5.8		4.7**
day 4		9.2			10.1	9.3		5.8**
Viability index		99.41			99.42	75.78*		11.93**
No. of external anomalies		0			0	0		0
	Gestation index = (Number of dams with live newborns/Number of pregnant females) x 100 Birth index = (Number of live newborns/Number of (stillborns + live newborns)) x 100 Viability index = [number of live newborns on day 4 after birth/number of live newborns] x 100 *: p < 0.05, **: p < 0.01 (significantly different from control) 1): All newborns were dead and counted as stillborn 2): All newborns were dead before day 4

Conclusions:

The parental NOAEL of reproductive toxicity was considered to be 100 mg/kg/day for males, 20 mg/kg/day for females and 4 mg/kg bw/day for breast feeding females based on the lack of nursing activity.

Executive summary:

In the OECD combined repeat dose and reproductive/ developmental (one generation)toxicity screening test [OECD TG 422], this substance was given for 49 days from 14 days before mating in males and from 14 days before mating to day 3 of lactation in females. At the 100 mg/kg bw group, one female died in gestation and another female was not impregnated. Birth index was decreased with increase in stillborns at the 100 mg/kg bw. All pups of two dams at the 20 mg/kg bw and seven dams at 100 mg/kg bw died due to the lack of nursing activity, and the viability index on day 4 after birth was consequently decreased in these groups. The body weights of pups were also decreased at birth and day 4 of lactation in the 100 mg/kg bw group. The decrease of litter size observed at the 100 mg/kg bw seems to be the chemical-induced effect although it is not statistically significant. No malformations or variations were observed in the pups.

In this study effects on birth index and body weight gain of newborns as reported. The NOAEL for these effects was set with 20mg/kg. The NOAEL for male rats was 10 mg/kg (highest dose applied). Female rats had a lower nursing activity for the group dosed 20 mg/kg bw and higher, the NOAEL for these effects was set with 4 mg/kg bw. The NOAEL for effects on F1 generation is closed to NOAEL from the 90 day study (19 mg/kg bw). The NOAEL for effects on nursing female rats (4 mg/kg bw) is lower than the NOAEL from the 90 day study (19 mg/kg bw). These results indicate that there might be a relevant potency for reproductive toxicity. It is not possible to finally evaluate, wether the effects on reproduction are the result of a generalized toxicity or wether the substance has a specific toxicity with respect to the endpoint.

The parental NOAEL of reproductive toxicity was considered to be 100 mg/kg/day for males, 20 mg/kg/day for females and 4 mg/kg bw/day for breast feeding females based on the lack of nursing activity.

Based on exposure considerations, the lower NOAEL (4 mg/kg bw/day) refering to the lack of breast feeding may not be taken into account for the assessment:

The substance is a monomer used for polymers and resins. RMM to minimise the exposure for the relevant subpopulation are taken.

Therefor the NOAEL (20 mg/kg bw/day) refering to the non-breast-feeding population is used for the risk characterisation.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

two studies are considered for this endpoint:
Izumi, 1997 and Szakonyine, 2022.
Both studies are conducted under GLP and are designed according
to OECD 421 or 422 (Screening studies).
In both studies effects were seen at mid doses of the respective study.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

Two studies on developmental toxicity according to OECD 414 are available.

The study by Kolep-Csete (2016) uses rats as test organisms. Developmental effects are seen which are observed, which are clearly related to the systemic toxicity of the substance and the resulting health status of the parental animals.

The OECD 414 study initiated by the registrant REACH24 (2022) uses rabbits as test organisms. Only limited information is available for this dossier, however this information is conclusive with the results from the study in rat:

Treatment of 16 to 22 pregnant New Zealand White rabbits per group from gestation day 6 to 27 by oral gavage administration of the test item 6-PHENYL-1,3,5-TRIAZINE-2,4-DIYLDIAMINE (BENZOGUANAMINE) formulated in the vehicle 0.5% aqueous Methylcellulose at dose levels of 5, 15 or 35 mg/kg bw/day caused no mortality.

The dose level of 35 mg/kg bw/day increased significantly the incidence and duration of less faeces (15 does), caused reduced water consumption of three animals, and in addition, no faeces (with no or minimal food consumption) as well as reduced activity and ascites as a consequence in one female. The test item caused no other finding revealed at necropsy.

The test item caused reduced food consumption, lower body weight/gain (including weight loss between G.D. 6 and 9) at 35 mg/kg bw/day. The reductions in food consumption and body weight gain in the 15 and 5 mg/kg bw/day dose group were considered as not adverse, based on the unaffected mean body weight values as well as the lack of dose response or lack of relationship between food consumption and body weight gain on the involved time periods.

The test item had no impact on the intrauterine parameters (intrauterine death of conceptuses, number of implantations and viable fetuses, or the distribution of the sexes).

6-PHENYL-1,3,5-TRIAZINE-2,4-DIYLDIAMINE (BENZOGUANAMINE) caused no malformations in the fetuses.

An impact of the test item, possibly due to maternal toxicity was revealed in the lower fetal weight (and not the length), a slight delay of ossification (less than 5 sternebra ossified) as well as relative placental weight (and not on the absolute value) at 35 mg/kg bw/day.

The test item had no adverse effect on fetal development observed by external and visceral examinations.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:

NOAEL maternal toxicity: 15 mg/kg bw/day
NOAEL developmental toxicity: 15 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start: 22 nd October 2015 End: 10th May 2016 (Start of experimental phase: 29 th October, 2015 - End experimental phase: 9 th December, 2015)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

Concerning: Number of sperm positive females, Concerning: Completion of section General Statements, Concerning: End of in-life phase

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

The test item was administered at appropriate concentrations, prepared with the vehicle. Preparation of the test item formulations was made with a frequency of 1 to 3 days, using a magnetic stirrer. Benzoguanamine in formulations was stored at room temperature for 4 hours or at 2-8 °C for maximum three days according to the results of the Validation of the Analytical Method for the Determination of Benzoguanamine Content (Study No: 849-100-0758). Sampling for analytical control of dosing was made on the first and last week of treatment. Concentrations of the test item in the dosing formulations at both analytical occasions was given in the Study Report

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals
Species / Strain: Hsd. Han: WIST Rats
Source: TOXI-COOP ZRT. 1103 Budapest, Cserkesz u. 90.
Hygienic level: SPF at arrival and kept in good conventional environment during the study
Number of animals: 130 females
80 males
Age of females at arrival: 8-9 weeks
Body weight of females at arrival: 160-180 g
Age of males at arrival: 8-9 weeks
Body weight of males at arrival: 230-260 g
Age at start of mating: females 9-10 weeks, males 9-10 weeks
Acclimatization time: 7 days

Environmental conditions
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 21-22 °C
Relative humidity: 32 - 51 %
Ventilation: above 10 air exchanges/hour by central air-conditioning system.

Environmental conditions were maintained by an air-condition system. Temperature and relative humidity were verified and recorded daily during the study.

Housing conditions
Animal health: Only healthy animals were used for the study. Health status was certified by the breeder
Animal room: 16/ A
Housing: pre-mating period: 1-3 females /cage,
2 males/cage
during mating hours: 1 male with 1- 3 females
during pregnancy: 2-3 sperm positive females /cage
Cage type: Type II polypropylene/polycarbonate with stainless steel covers equipped by self-feeding baskets
Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-7349 4 Rosenberg Holzmühle 1 Germany). The cages and bedding were changed twice a week.
Room sanitation: At the end of each working day floors were swept and then mopped with an acceptable disinfectant. Water bottles were cleaned on a rota basis as required during the course of the study.

Food and water supply
Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum. Contents of standard diet for rats and mice are presented in Appendix XXII/A.
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used.
Water quality control analysis was performed periodically. The quality control results are available at Toxi-Coop Zrt’s archives.

Route of administration:

oral: gavage

Vehicle:

other: Methylcellulosum (Ph. Eur. 7.) and Distilled water (Aqua purificata Ph.Hg. VIII)

Remarks on MMAD:

According to particle size determination, 54 % of the particle are above 125 µm.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated HPLC/UV method. The measured concentrations of the Benzoguanamine formulations varied between 102 and 119 percent of the nominal concentration.

Details on mating procedure:

The females were paired to males in the mornings for two to four hours (one male: one to three females) until the number of sperm positive females / group achieved at least twenty two and the anticipatory number of litters at Caesarean section achieves at least 16.
Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle. The day of mating was regarded as day 0 of pregnancy (vaginal plug and/or sperm in the vaginal smear). Sperm positive females were separated and caged in groups of 1 to 3 animals individual caging was avoided.
Randomization
The sperm positive females were allocated if possible to each experimental group on each mating day in such a way that the group averages of the body weight were as similar as possible on the first day of gestation. Females paired with the same male were allocated to different groups on the same mating day.

Duration of treatment / exposure:

Groups of 34, 30, 27 and 36 sperm-positive female Hsd. Brl. Han: WIST Rats were treated with Benzoguanamine by oral administration at three dose levels of 25, 50 and 125 mg/kg bw/day and one control group from day 5 up to and including day 19 post coitum daily. The control animals were given the vehicle (0.5% methylcellulose) alone.

Frequency of treatment:

Daily

Duration of test:

Treatment period from 3th November 2015 to 8 th December 2015

Dose / conc.:

25 mg/kg bw/day

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

No. of animals per sex per dose:

pre-mating period: 1-3 females /cage,
2 males/cage
during mating hours: 1 male with 1- 3 females


Dose Number of
Group (mg/kg/bw/day) sperm positive females
Control 0 34
Low 25 30
Mid 50 27
High 125 36

Control animals:

yes

Maternal examinations:

Clinical observation
General clinical observations of the sperm positive females were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. When signs of toxicity were observed, animals were checked more frequently.
Individual observation included a check of behavior and general condition.
Duration and severity of the clinical signs were recorded.

Mortality
Observations for signs of morbidity and for mortality were made twice daily, at the beginning and before the end of the working period.

Body Weight

The body weight of the male animals was not measured.

The body weight of the female rats was measured at least once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).
Corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus).

Food Consumption

The food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).


Examination for Sign of Implantation

On gestation days 13 and/or 14 the sperm positive females were checked for the presence of vaginal bleeding which indicated the implantation of conceptuses

Ovaries and uterine content:

All sperm positive females were sacrificed by decapitation under deep Isofluran anaesthesia on day 20 of gestation. The abdomen was opened, the uterus with cervix and left ovary were removed and weighed. The right ovary was placed into a Petri dish after removal. After removing the uterus gross pathology of dams' viscera was performed.
The number of corpora lutea in each ovary and implantation sites in each uterine horn, live fetuses, early and late embryonic death and fetal death were counted. Animals in which unambiguous implantation sites, but not fetuses, were found were considered as pregnant. Uteri that appear non-gravid were further examined to confirm the non-pregnant status.

Fetal examinations:

Fetuses were removed from the opened uterus and were sunk in a Petri-dish filled up with water. Spontaneous movement of fetuses was observed as a viability assessment. Euthanasia of the fetuses was performed by hypothermia.
The fetuses were washed with tap water and randomly laid on a filter paper with written ordinal numbering. Bleeding from the umbilical cord after it is cut was observed as an indication of viability before euthanasia.
Each live fetus and its placenta was weighed individually (fetuses accuracy 0.01 g, placentas accuracy 0.001 g), and subjected to external examination. The gender of the fetuses was determined according to the anogenital distance. The fetuses were individually identified and about the half of each litter was subjected to visceral examination and the other half for skeletal examination. The body of those subjected to visceral examination was fixed in Sanomiya mixture. After fixation the bodies were micro dissected by means of a dissecting microscope.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Vaginal bleeding was observed in one female in the 125 mg/kg bw/day group. Considering the low incidence, this was not attributed to the treatment.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the in-life phase

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight of the females was statistically significantly higher (p<0.01) in the 125 mg/kg bw/day dose group before the treatment period, the body weight gain was at similar level as the control.

During the treatment period, from gestation day 11 up to the end of the in-life phase the body weight of the dams in the 125 mg/kg bw/day group was statistically significantly lower (p<0.01) versus control. Lower body weight was measured also in the 50 mg/kg bw/day dose group on gestation day 14 (p<0.05) and on gestational day 17 and 20 (p<0.01). There was no significant difference in the body weight of the dams in the 25 mg/kg bw/day group versus control.

Weight loss from gestation day 5 to 8 and reduced body weight gain (marked between days 8 and 11 and if calculated from 0 to 20 (p<0.01) and moderate between days 11 to 14 and 17 to 20 (p<0.05 and p<0.01 respectively) was indicated in the 125 mg/kg bw/day dose group. The body weight gain was moderately reduced in the 50 mg/kg bw/day group during the whole treatment period (p<0.05 and p<0.01).
The corrected body weight parameters also reduced with a dose response in the 50 and 125 mg/kg bw/day dose groups.
The reductions in the body weight parameters of the dams in the 50 and 125 mg/kg bw/day dose group were attributed to the treatment.
Slightly lower and statistically significant body weight gain was calculated for the dams in the 25 mg/kg bw/day dose group between days 5-8 and 8-11 (p<0.05 and p<0.01 respectively) as well as if calculated for 0 and 20 (p<0.05). There was no significant difference in the corrected body weight and corrected body weight gain of the dams in this dose group if compared to the control.
The statistically significantly lower body weight gain of the females in the 25 mg/kg bw/day dose group was not clearly attributed to the treatment and judged as non-adverse considering the slight degree and also that the corrected body weight of the dams was similar as the current control values.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The food consumption of the dams in the 125 mg/kg bw/day group reduced significantly (-34%, p<0.01) on the first three days of treatment, became moderate (-23%, p<0.01) from gestational day 8 to 11 and slight (-13% (p<0.01) between days 11 and 14.
The reductions in the food consumption from gestation day 5 to 11 were considered to be due to the treatment with the test item. The lower value between gestation days 11 and 14 was judged to be not adverse considering the slight degree.

Statistical significance (p<0.01) was indicated in the 50 (-19 and -11%) and 25 (-12 and
-9%) mg/kg bw/day groups between gestation days 5-8 and 8-11 respectively for the slightly lower food consumption values. The moderate reduction of the food consumption in the 50 mg/kg bw/day group between days 5-8 was likely caused by the treatment. The 11% decrease between days 8-11 was not clearly attributed to the treatment and was judged as non-adverse.
The lower values were considered to be likely not related to the treatment and non-adverse in the 25 mg/kg bw/day group considering the slight degree. Moreover a statistically significant (p<0.05) increase was to see between days 14 and 17 in this group.

Statistically significant increases (p<0.05 and p<0.01) were also indicated in the pre-treatment period in the 125 mg/kg bw/day group group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clotted blood in the uterine horn was recorded for one dam in the 125 mg/kg bw/day dose group. Considering that one female in the control group had similar macroscopic finding at necropsy (blood in the uterus) this change was considered as to be without a relationship to the treatment. There were no macroscopic changes recorded for the females in the 25 and 50 mg/kg bw/day dose groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

postimplantation loss was statistically significantly higher due to the higher early (p<0.01) and late (p<0.05) embryonic death according to the statistical analysis with CH square (Appendix VI/B). However there was no statistically significant difference in the mean percent of these parameters in the dose groups versus control (Appendix VI/A), moreover the values did not exceed the range in the background database (XXIV/A, B). This suggests that the increase in the postimplantation loss in the 125 mg/kg bw/day in the present study could be unrelated from the treatment.
There was no significant increase of early- and late embryonic death and postimplantation loss in the 25 and 50 mg/kg bw/day dose groups if compared to the control. The number of total death due to the preimplantation loss was statistically significantly higher p<0.05 in the 25 mg/kg bw/day dose group but without a dose response.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

There was one single dead fetus in the control group found at examination of the uteri.

Changes in pregnancy duration:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight of the fetuses in the 50 and 125 mg/kg bw/day dose groups was lower (p<0.01) with a dose response which was attributed to the treatment of the dams. There was no statistical significant difference indicated in the mean body weight of the fetuses in the 25 mg/kg bw/day dose group vesus control.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two fetuses were found with a malformation (microphthalmy) in the 25 mg/kg bw/day group. Considering that microphthalmy occurs sporadically with low incidence unrelated to the treatment according to the experience with this species in this laboratory and to the historical control data and in line with historical control data of another strain of rats, this was judged not to be a consequence of the treatment of the dams with Benzoguanamine.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Dumb-bell shaped thoracic centrum cartilage recorded for two fetuses in the control group and in one in the 25 mg/kg bw/day group was judged to be unrelated from the treatment.
Split sternum found in one fetus in the 25 mg/kg bw/day (the same as with umbilical hernia) and in three in the 125 mg/kg bw/day dose group was judged to be unrelated from the treatment based on the Historical Control Data and the experience with this species in this laboratory which is in line with historical control data of another strain of rats by which split sternum may occur without any test item relationship with a low incidence, moreover the male mated with two of these dams was the same which further reduced the probability of a test item effect.

The incidence of skeletal variations increased significantly in the 125 mg/kg bw/day dose group as a consequence of the treatment.
Statistically significantly increased the incidence of bipartite supra occipital bone, 3 or less ossified sternebra and less than 3/3.5 ossified metacarpal/metatarsal in the 125 mg/kg bw/day dose group in accordance with the markedly low fetal weight. There was no statistical significance indicated in the increase of variations in the other groups if evaluated on the basis of litter incidences.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

In addition to the fetus with umbilical hernia in the 125 mg/kg bw/day dose group which was allocated to visceral examination

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Conclusions:

Based upon these data, treatment of pregnant Hsd. Han: WIST Rats from gestational day 5 to 19 by oral administration of Benzoguanamine, caused maternal toxicity such as reduced body weight and body weight gain at the dose level of 125 and 50 mg/kg bw/day (including weight loss at 125 mg/kg bw/day) and reduced food consumption. The slightly reduced food consumption at 25 mg/kg bw/day and slightly lower body weight gain at 25 mg/kg bw/day was not clearly attributed to the treatment and was considered as non-adverse.
Benzoguanamine caused no clinical signs and adverse necropsy changes of the dams. The treatment of the dams with test item was judged not to result in increased intrauterine mortality and fetal malformations. Benzoguanamine caused dose related lower fetal weight and higher incidence of body weight retardation (evaluated as external variation) at 125 and 50 mg/kg bw/day as well as increased incidence of skeletal variations in association with the lower fetal weights in the 125 mg/kg bw/day dose group.

Based on these observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL maternal toxicity: 25 mg/kg bw/day
NOAEL developmental toxicity: 25 mg/kg bw/day

Executive summary:

Groups of 34, 30, 27 and 36 sperm-positive female Hsd. Brl. Han: WIST Rats were treated with Benzoguanamine by oral administration at three dose levels of 25, 50 and 125 mg/kg bw/day and one control group from day 5 up to and including day 19 post coitum daily. The control animals were given the vehicle (0.5% methylcellulose) alone. The treatment volume was 5 mL/kg/bw.

Formulation analytics (checking of homogeneity and achieved concentrations of the test item in the dosage forms) was performed two times during the treatment period using a validated HPLC/UV method. The measured concentrations of the Benzoguanamine formulations varied between 102 and 119 percent of the nominal concentration. During the study animals were checked for mortality and clinical signs. Body weight and food consumption of the dams were also recorded. The day of detection of sperm in the vaginal smear of females was regarded as day 0 of gestation. A Caesarean section and gross pathology were performed on gestational day 20. Organs of the dams were examined macroscopically.

The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The placentas were weighed and examined externally.The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method.After double staining, the skeletons were examined by means of a dissecting microscope.All abnormalities found during the fetal examinations were recorded.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

7

Species:

rat

Quality of whole database:

Two studies are available covering this endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

a mode of action could not be identified.

The general, systemic toxicity may be the major reason for the observed effects, however, agalactia and effects on male sexual organs have to be seen as additional toxic actions, caused by the substance.

As systemic toxicity is seen in two different animal species, a species selective mechanism can be excluded and the effects may be relevant for humans as well.

Justification for classification or non-classification

The data is conclusive and sufficient for classification as Reproductive toxic cat. 1B