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

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

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:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Benzoguanamine
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.7 %
- Impurities (identity and concentrations): -
- Composition of test material, percentage of components: -
- Isomers composition: -
- Purity test date: -
- Lot/batch No.: 404502
- Expiration date of the lot/batch: 01.01.19
- Radiochemical purity (if radiolabelling): 99.8 %
- Specific activity (if radiolabelling): 58 mCi/mmol (11.35 MBq/mg or 681000 dpm/µg)
- Locations of the label (if radiolabelling): s. background material
- Expiration date of radiochemical substance (if radiolabelling): -
- Stability under test conditions: stable
- Storage condition of test material: - 20 °C
- Other: -

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

Test animals

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.

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

25 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day

Doses / concentrations 3

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

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

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

Results (fetuses)

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

Effect levels (fetuses)

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

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

not specified

Applicant's summary and conclusion

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.