4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

EC Number:

276-309-1

EC Name:

4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine

Cas Number:

72058-41-4

Molecular formula:

C11H18Cl2N4

IUPAC Name:

4,6-dichloro-N-(2,4,4-trimethylpentan-2-yl)-1,3,5-triazin-2-amine

Test material form:

solid

Details on test material:

Colour: light yellow

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SUQIAN UNITECH CO., LTD; Unitechem-20211231
- Purity: 99.29%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

other: Wistar Crl: WI(Han) (Full Barrier)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: Age of the females at the arrival at the test facility - 11-12 weeks old; Age of the males at the start of pairing - 12-13 weeks old.
- Weight at study initiation: males: 322 - 419 g (mean: 358.07 g, ± 20% = 286.45 – 429.68 g); females: 194 - 248 g (mean: 222.76 g, ± 20% = 178.21 – 267.31 g)
- Housing: individually in IVCs (type III H, polysulphone cages) on Altromin saw fibre bedding (except during the pre-mating period when females were kept in groups of two animals and during mating period when two females were paired with one male). The pregnant females were provided with nesting material towards the end of the pregnancy (e.g. at GD 18)
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 40–70%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The purpose of the formulation study was the quantification of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine in samples received from the BSL Munich Study No. 2200010 for verification of concentration and homogeneity using a HPLC-UV method.

Prior to sample analysis, the method was revalidated to ensure the validity of the validated method (Eurofins Munich Study No. 183815 (GLP), and was confirmed as adequate. Stability was confirmed in this previous study.

Concentration Analysis: Concentration analysis of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 55 mg/mL in study week 1 and 10 mg/mL, 30 mg/mL and 45 mg/mL in the last week of the study. The mean recoveries observed for the LD group was between 90.4% and 101.3% of the nominal value, between 88.1% and 94.5% for the MD group and between 92.5% and 96.0% of the nominal value for high dose (HD) group. The mean recoveries observed in the LD, MD and HD groups were 95.9%, 91.3%, and 94.4% of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured mean concentrations were within acceptance criterion of 10%.

Homogeneity: Homogeneity of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 55 mg/mL in study week 1 and 10 mg/mL, 30 mg/mL and 45 mg/mL in the last week of the study. The coefficients of variation of the different sampling locations (top, middle, bottom) were between 0.2% and 1.0% in LD group, 0.2% in MD group and between 0.3% and 2.2% in HD group. All samples were homogenous, as COV was below or equal 10%.

Details on mating procedure:

Mating was performed using a ratio of 1:2 (male to female). Females were paired for cohabitation in batches in order to control the number of animals for terminal sacrifice on a particular day. At the subsequent mornings, the vaginal smear of the females was checked to confirm the pregnancy. The day on which sperms are observed in the vaginal smear is considered as “GD 0”. Mated females were assigned in an unbiased manner to the control and treatment groups ensuring that group mean body weights are comparable with each other. Each animal was assigned a unique identification number. After getting 92 sperm-positive females, the remaining females and males were discarded without any observation.

Duration of treatment / exposure:

GD 5 - GD 19

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low Dose (LD)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Mid Dose (MD)

Doses / concentrations 4

Dose / conc.:

275 mg/kg bw/day (nominal)

Remarks:

Dose was reduced due to severe clinical signs and morbidity in the HD group; (HD group treated at 225 mg/kg/day - Animal no. 70 was dosed from GD16, Animal no. 73 from GD 10, Animal no's 74/75 from GD7 and Animal no. 76-78 from GD6)

No. of animals per sex per dose:

23 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Refer to Supporting, RL1, rat/Suqian, 2022/Developmental toxicity / teratogenicity.002

Examinations

Maternal examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

BODY WEIGHT: Yes
All animals were weighed once before initiation of pairing to ensure that the body weights are within ±20% variation. The sperm-positive females were weighed on GD 0, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study except once before initiation of pairing.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
Food consumption of sperm-positive females was determined for the following intervals: GD 0 5, 5 8, 8 11, 11 14, 14 17 and 17 20. Food consumption was not measured for males during the entire study or for both males and females during the mating period.

POST-MORTEM EXAMINATIONS: Yes
On GD 20, sperm-positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using anaesthesia (ketamine/xylazin).
At the time of termination or death during the study, each dam (presumed pregnant female) was examined macroscopically for any structural abnormalities or pathological changes which may have influenced the pregnancy. Any macroscopic findings were preserved in 4% neutral-buffered formaldehyde.

Thyroid/parathyroid glands from all dams were preserved in 4% neutral-buffered formaldehyde. The weight of thyroid/parathyroid glands was measured after 24 hours fixation. A histopathological evaluation was carried out on the preserved thyroid/parathyroid glands from all dams of all dose groups sacrificed at the end of the treatment period.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes

Immediately after the termination or as soon as possible after death, the uteri were removed and the pregnancy status of the dams was confirmed. Uteri that appeared non-gravid were further examined by staining with 10% ammonium sulphide solution to confirm the non-pregnant status.
Each gravid uterus with cervix was weighed. However, the gravid uterus obtained from dead animals was not weighed.

The number of corpora lutea was counted for pregnant animals. The uterine contents were examined for embryonic or foetal deaths and the number of viable foetuses. The degree of resorption (late and early) was confirmed in order to help estimate the relative time of death of the conceptus. The position and number of foetuses in each uterine horn was recorded.

Blood sampling:

Thyroid hormone levels from samples from all dams were assessed at the end of treatment prior to or as part of the sacrifice of the animals. At termination, blood samples were collected from the defined site in serum separator tubes and obtained serum was stored under appropriate conditions. Serum samples were assessed for thyroid hormone levels (T3, T4, TSH) using ELISA.

Fetal examinations:

All foetuses from a particular dam were identified using numbered plates and were weighed and sexed based on the anogenital distance (AGD). Each foetus was examined for external anomalies and the AGD of each foetus was measured. Foetal body weight measured on GD 20 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD / cube root of foetus weight). Particular attention was paid to the reproductive tract which was examined for signs of altered development. External foetal sex (as determined by gross examination) was compared with internal (gonadal) sex in all foetuses (examined for both skeletal and soft tissue malformations). In addition, indication of incomplete testicular descent/cryptorchidism was noted in male foetuses.

External Examination: One half of each litter was examined for soft tissue anomalies of the body by a microdissection technique. Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout was examined for the shape and size. The trunk was examined for any external abnormalities. Limbs were examined for shape, size, position and digits for number and depth of digital furrows. The tail was examined for presence, size, shape and position.


Visceral Examination
One half of each litter was examined for soft tissue anomalies of the body by a microdissection technique. After the completion of the external examination, foetuses were transferred to plastic bottles containing Bouin’s solution for later visceral and craniofacial examination. The visceral evaluation of the body and craniofacial examination (eyes, brain, nasal passage and tongue by razor blade serial sectioning technique) of the heads of the same foetuses of at least 20 litters per group was performed.
The intestine, stomach, spleen and pancreas were examined for size and position. The liver was examined for size, shape, colour and number of lobes. The kidney and adrenal glands were observed for size, position and colour. The reproductive organs were exposed by raising the intestine and the attached viscera from the dorsal wall and examined for any developmental defect.
The rib cage was cut from the side of the sternebrae and xyphisternum (6th sternebra) to examine the thoracic organs. The lung was observed for size, colour and number of lobes. The thymus gland was checked for size and position. The trachea and oesophagus were exposed by removing the thymus gland and examined for fusion or tracheaoesophageal fistula.
The position, size, colour and shape of the heart were recorded. The pericardial sac was opened and the heart was fully exposed and examined for the presence or absence of major blood vessels like aortic arch, pulmonary artery and ductus arteriosus.

Craniofacial Examination
After deformalisation, a single foetus was decapitated and the head of the foetus was subjected to 5-7 sections in order to observe the internal structures of the head including the symmetry of the external nares, nasal conche, nasal septum, palate, the development of the cerebellum and brain stem. Transverse sections of the cephalic region were observed under the stereomicroscope and any anomalies were recorded.


Skeletal Examination
Foetuses scheduled for the skeletal examination were eviscerated and the entire litter was transferred into plastic bottles containing 95% ethanol. These foetuses were processed using the Alizarin red staining technique.
The stained foetuses were examined under the stereomicroscope, the skull was examined for size, shape and degree of ossification of nasal, parietal, interparietal, supraoccipital, exoccipital, lacrimal, zygomatic (malar), squamosal (temporal), premaxillary, maxillary, basisphenoid, hyoid and tympanic ring (annulus). Similarly, the vertebral centres, ribs and sterna centres were also examined for size, shape and counted for the number of ossification centres. The cervical, thoracic, lumbar, sacral, caudal vertebrae were observed for the ossification of centres and arches. Pelvic girdles, fore limbs and hind limbs were examined for the development of the bones. Any deviation from the normal development was recorded for each foetus.

Statistics:

A statistical assessment of the results of the body weight and food consumption was performed by comparing values of dosed animals with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, thyroid hormones and foetal evaluation parameters like external, visceral, craniofacial and skeletal parameters were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. The statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 is considered as statistically significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Both HD dams who died showed severe clinical signs of toxicity which includes showed reduced spontaneous activity, hunched posture, dehydration, piloerection, both eyelids closed, abnormal breathing, nasal discharge, dehydration and increased salivation. After reduction of HD from 275 to 225 mg/kg bw/day, the severity of clinical signs was reduced and all other dams showed clinical signs of salivation, piloerection and moving the bedding materials on different gestation days. Test item-related clinical signs were observed in all the treatment groups in a dose dependent manner. Salivation, piloerection and moving the bedding materials were observed in the treated groups were considered to be local reaction to the treatment with test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Test item-related mortality was observed in the HD group (2 non-pregnant dams) treated at 275 mg/kg bw/day on GD 12 and 11 respectively. One dam was found dead in LD group (no. 45) on GD 14 which was assumed to be incidental (gavage error).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weight was affected by the test item and decreased with the progress of the study in MD and HD groups. Statistically significant lowered mean body weight was observed in MD and HD groups on GDs 8, 11, 14, 14 and 20 compared to control. Similarly, statistically significant lowered mean body weight gain was observed in MD and HD groups on GDs 5-8, 8-11, 17-20 (only in HD group). The reduction in mean body weight gain was observed in MD and HD groups on GDs 5-20 (30.22%, 42.82% respectively) of treatment days compared to control. The mean body weight and body weight gain was found to be comparable to control in LD group during the treatment periods. No test item-related effects were observed on GDs 5-20 in LD group and comparable to control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In correlation to lowered mean body weight or gain in MD and HD groups, test item-related effect and statistically significant changes in mean food consumption was observed in MD and MD groups during the treatment days of gestation days. The mean food consumption of the MD and HD groups were lowered on GDs 5-20 with statistical significance (22.43% and 27.54% respectively) when compared to control. Changes observed in MD and HD groups are considered to be test item-related and dose dependent. The mean food consumption of LD group was found to be comparable to control during GDs 5-20.

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

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related effects were observed on group mean T3, T4 and TSH hormone levels and values were comparable to the control group. No test item-related changes were observed in anogenital distance (AGD) in the treated groups.

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

Description (incidence and severity):

The test item did not show any toxicologically significant changes in mean uterus weight, however the mean carcass weight was lowered in MD and HD groups with statistical significance (10.10% and 13.17% respectively). The lowered carcass weight in MD and HD groups are considered to be test item-related effects.

Statistical analysis of post-fixed thyroid/parathyroid weights from all dams revealed no statistically significant or toxicologically relevant effect on the absolute and relative (to body weight) thyroid/parathyroid weights of the dose groups when compared to the control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related macroscopic observation were observed in MD and HD groups. The test item-related changes were observed predominantly in kidneys, including abnormal coloured (pale, white, pink, dark red), spotted pelvis, enlarged, abnormal surface (rough, vesicular), abnormal consistency (soft). Other findings in HD group were liver (dark, marbled, spotted) in 3 animals and 2 animals in MD group. Enlarged spleen was observed in one animal in the MD and HD groups. Abnormal coloured, pale lungs in one animal in the LD group and pale kidneys in one LD animal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no test item-related findings at histopathological evaluation of the thyroid and parathyroid glands in any of the treated groups observed.

The substance was classified as STOT-RE 2 (oral, kidney) based on the previous OECD 422 study so no further histopathological investigations were performed on other organs.

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

gross pathology

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean male and female foetal weights observed on an individual basis (sum of weight of all foetuses in group divided by total number of foetuses in respective group) in the MD (Male: 8.03%, Female: 7.32%) and HD (Male: 12.99%, Female: 14.83%) groups were slightly reduced when comparable to control in a dose dependent manner but without any statistically significance. The mean foetal weights of LD group were found to be comparable to control.
A statistically significant decrease in male and female mean foetuses weight of MD (3.58 g Vs 3.85 g and 3.35 g Vs 3.63 g) and HD (3.30 g Vs 3.85 g and 3.13 g Vs 3.63 g) groups were observed when compared to control, with dose dependent effects. The mean foetal weights of LD group were found to be comparable to control (male and female foetal weight per litter basis).

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, the relative anogenital distance (AGD) in the treated groups was not affected by the test item and no statistically significant changes were observed in LD and MD groups when compared to the control. In the HD group, the AGD was statistically significantly lowered (2.541 mm vs 2.599 mm) and relative AGD was slightly higher in HD groups (1.703 mm vs 1663 mm) when compared to control group, however these findings were observed without any dose dependency and in addition, all these values are within the historical control range of this strain; hence it is not considered to be test item-related effects.

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related external abnormalities observed in any of the foetuses of treated groups. However, slightly higher litter incidences of small foetuses were observed in MD (19%) and HD (32%) groups when compared to control (5%) without any statistical significance changes. This could be due to secondary effects of maternal toxicity in terms of reduced mean body weight and food consumption. The other observed changes in few foetuses were considered to be incidental in nature.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically significantly higher litter incidences of 1st sternebra incomplete ossification was observed in HD group (38.9% vs 5% control) which is slightly higher than historical control data (25%) on litter basis. Statistically significantly higher foetal incidences of 2nd sternebra (incomplete ossification and unossified) was observed in HD group (48.41%) when compared to control (24.52%). Statistically significantly higher total foetal incidences of 5th sternebra was observed in HD group (66.86%) when compared to control (39.26%). Statistically significant lower litter incidences of skull parietal incomplete ossification were observed in HD group (22.2%) when compared to control (65.0%). Statistically significantly higher foetal incidences of skull supraoccipital (incomplete ossification and with small hole) was observed in HD group (95.24%) when compared to control (79.79%). Statistically significant higher foetal incidences of skull supraoccipital incomplete ossification was in HD group (92.06% vs 73.12% in control) which is slightly higher than historical control data (90.8%) on fetal incidence basis. Statistically significant higher foetal incidences of skull supraoccipital with hole was in MD group (47.15%) when compared to control (20.64%).
Statistically significantly lower foetal incidences of skull parietal incomplete ossification were observed in MD group (0.83%) when compared to control (7.02%). Statistically significantly higher foetal incidences of 14th rib (L) was observed in LD (24.36%) and MD groups (24.70%) when compared to control (11.24%). Statistically significantly lower foetal incidences of vertebra sacral, fused were observed in HD group (0%) when compared to control (11.74%). Statistically significantly lower foetal incidences of vertebra caudal, fused were observed in HD group (0%) when compared to control (8.19%) and lower litter incidence in HD group (0%) when compared to control (35%). All these findings were observed without dose dependency or consistency; hence they are not considered to be test item-related.
Other skeletal findings were observed without achieving statistical significance or dose dependency. All the observed skeletal changes were within normal biological variations.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No statistical significance was observed in any of the treated groups when compared to the control group. There were higher or lower litter incidences of umbilical artery malpositioned in the LD, MD and HD groups (60%, 40% and 47% respectively) when compared to 50% in control. Abdomen, internal haemorrhage was observed in the LD, MD and HD groups (15%, 25% and 16% respectively) when compared to 20% in control. Higher incidence of supernumerary liver lobe was observed in LD (20%) and HD (15.8%) groups when compared to control group (0%). Lower litter incidences of testis, malpositioned was observed in the LD, MD and HD groups (15%, 10% and 11%, respectively) when compared to 25% in the control. Higher incidence of long thymus was observed in MD (15%) when compared to control (10%).
The observed findings were found to be either minor variations and/or due to a lack of dose dependency and consistency, no toxicological significance can be attributed to these findings and they were considered to be spontaneous in nature.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed slightly higher litter incidences of subcutaneous oedema (head) was observed in HD group (16.7%) when compared to control (15%). Increased meningeal space was observed slightly lower in LD group (15%) when compared to control (25%). Lens, altered texture was observed slightly lower in LD group (25%) when compared to control (30%). All these findings were considered to be spontaneous in nature and not related to test item. Statistical analysis of the data revealed no statistical significance in any of the treated groups when compared to control.

Details on embryotoxic / teratogenic effects:

Test item-related reduction in mean foetal weight (individual and litter basis) were observed in MD and HD groups; which could be due to the secondary effect of maternal toxicity with reduced food consumption and body weight. No test item-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of all treatment groups when compared to control group.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

In a prenatal developmental toxicity in Wistar Crl: WI(Han) rats, the NOAEL for maternal toxicity of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was considered to be 50 mg/kg bw/day, based on the test-item related gross pathology lesions in the kidneys. The NOAEL for foetal toxicity of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was considered to be 50 mg/kg bw/day, based on the test-item related reductions in mean foetal weights.

Executive summary:

In a prenatal developmental toxicity in rats (OECD 414/GLP), the test item (99.29%) was administered to pregnant Wistar, Crl: WI(Han) rats (23/dose) by oral gavage in corn oil at dose levels of 0, 50 (LD), 150 (MD) and 275/225 (HD) mg/kg bw/day daily from GD 5-19.

Concentration analysis of formulation samples indicated mean recoveries in the LD, MD and HD groups were 95.9%, 91.3%, and 94.4% of the nominal concentration, respectively. All samples were homogenous.

Test item-related mortality was observed in the HD group (2 non-pregnant dams) and no further morbidity was observed after reduction of the dose to 225 mg/kg bw/day. Clinical signs of toxicity were observed in all treatment groups during the study period in a dose dependent manner; severity of clinical signs predominated in the HD group. One dam was found dead in LD group (on GD 11 which was assumed to be incidental (gavage error). Test item-related effects on mean body weight, mean body weight gain, terminal body weight, adjusted maternal weight (carcass weight) and mean food consumption were observed in MD and HD groups.

Test item-related effects on gross pathology lesions were observed in MD and HD groups, predominantly in the kidneys. No test item-related effect was observed on group mean T3, T4 and TSH hormone levels and thyroid/parathyroid weights from all dams. No test item-related findings at histopathological evaluation of the thyroid gland in any of the treatment groups.

Successful mating resulted in 21/23 pregnancies in the LD group, 21/23 in the MD group and 19/23 in the HD group compared to 22/23 pregnancies in the control group. Test item-related reduction in mean foetal weight (individual and litter basis) were observed in MD and HD groups; which could be due to the secondary effect of maternal toxicity with reduced food consumption and body weight. No test item-related effects were observed on prenatal parameters including uterine weight, number of corpora lutea, implantation sites, early and late resorptions, percent pre and post implantation loss, number of live foetuses, anogenital distance (AGD), number of male and female foetuses, sex ratios and testicular descent in treatment groups when compared to the control. No test item-related and toxicologically relevant external, visceral, craniofacial and skeletal findings were observed in the foetuses of all treatment groups when compared to the control group.

The NOAEL for maternal toxicity of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was considered to be 50 mg/kg bw/day, based on the test-item related gross pathology lesions in the kidneys.

The NOAEL for foetal toxicity of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine was considered to be 50 mg/kg bw/day, based on the test-item related reductions in mean foetal weights.