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

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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.

Test material

Test material information

impurity 1

Reference substance name:

unknown impurities

Molecular formula:

not applicable

IUPAC Name:

unknown impurities

Constituent 1

Reference substance name:

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

EC Number:

202-095-6

EC Name:

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

Cas Number:

91-76-9

Molecular formula:

C9H9N5

IUPAC Name:

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

Test material form:

other: powder

Specific details on test material used for the study:

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

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

Overall reproductive toxicity

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

Applicant's summary and conclusion

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.