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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Toxicity to Reproduction (Screening):

Subacute NOAEL (reproduction, parental): 120 mg/kg bw/day (OECD 422/GLP)

Subacute NOAEL (development, offspring): 120 mg/kg bw/day (OECD 422/GLP)

 

 

Extended one-generation reproductive toxicity:

Results from the OECD 422/GLP and OECD 414/GLP studies (RL1) shows no adverse effects on reproductive organs or tissues in males or females, so the extended one-generation reproductive toxicity study (OECD 443) is waived.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 November 2018 - 26 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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; 2018041002
- Purity:≥ 99.29 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: stable in the corn oil

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item formulations were prepared freshly on each administration day before the administration procedure. The test item were emulsified in corn oil.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

healthy Wistar rats, Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males: 14 - 15 weeks old, females: 14 - 15 weeks old.
- Weight at study initiation:males: 330 - 369 g(mean: 347.55 g, ± 20 % = 278.04 – 417.06 g)
females: 204 - 250 g (mean: 223.38 g, ± 20 % = 178.70 – 268.05 g)
- Housing: Full barrier in an air-conditioned room; -Animals were housed in groups of 5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males depending on the mating status. During mating period males and females were housed together in ratio 1:1 (male to female). After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages and males were returned to their original cage. In each cage Altromin saw fibre was used as bedding. Nesting material were provided latest on GD 18 for all mated females-
- 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 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulation was prepared with corn oil. The vehicle was selected based on the test item’s characteristics and testing guideline.
Based on the results of stability testing (183815; Appendix 4), the test item formulations were prepared freshly at least once every 11 days (within stability time frame as given by Eurofins Munich Study No. 183815). The test item was weighed into a tared plastic vial on a precision balance. The dose formulations were prepared by adding the required volume of corn oil to give the appropriate final concentration of the test item. The formulation was homogenized by subjecting the formulation to an ultrasonic bath (40 °C) until visual homogeneity was achieved (at least 30 min). Formulates were kept under magnetic stirring during the daily administration. The vehicle was also used as control item.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item does not make a solution or suspension with water, so the corn oil has to be used.
- Amount of vehicle (if gavage): 4 mL/kg body weight
- Lot/batch no. (if required): MKCD1021 / MKCG3257

Details on mating procedure:

Mating was performed using a ratio of 1:1 (male to female) (if possible). The vaginal smear of the females was checked every morning after the start of the mating period to confirm the mating. If the vaginal smear of a particular female was not found to be sperm-positive, the actual stage of the estrus cycle on that day was documented. The day of the vaginal plug and/or sperm was considered as day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before beginning of the treatment period, formulation samples were prepared and analysed in order to obtain knowledge about stability and homogeneity of the test item in the selected vehicle at Eurofins Munich as part of a separate GLP study (183815; Appendix 4).

Prestart homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups.
The test item was shown to be homogenous according to 183815 (Appendix 4). However, as the test item forms a dispersion in the vehicle, samples will be collected during the study for the investigation of homogeneity and substance concentration. Samples will be taken from the top, middle and bottom of prepared formulations from all dose groups and from the middle of the control group in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) (40 samples).

Concentration Analysis
Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 3 mL). The A-samples were analysed at Eurofins Munich (Eurofins Munich Study Phase No. 183816) and until then stored under appropriate conditions based on available stability data. Concentration analysis of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 90/60 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The concentration of the HD dose group was reduced at the end of week 1. The mean recoveries observed for the LD dose group was between 99.7 % and 103.9 % of the nominal value, between 100.2 % and 102.2 % for the MD dose group and between 98.6 % and 102.5 % of the nominal value for HD dose group. The mean recoveries observed in the low dose (LD), medium dose (MD) and high dose (HD) groups were 101.8 %, 100.9 %, and 100.5 % of the nominal concentration, respectively. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 % (Appendix 4)

Homogeneity
Homogeneity of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 90/60 mg/mL, in study weeks 1, 3, 5 and the last week of the study. The coefficients of variation of the different sampling locations (top, middle, bottom) was between 0.3% and 1.3% in LD dose group, between 0.2% and 0.9% in MD dose group and between 0.3% and 1.4% in HD dose group. All samples were homogenous, as COV was below or equal 10%.

Note: Method validation is presented in 183815 (Appendix 4). Before the last sample measurement the method was transferred to a different HPLC instrument, due to maintenance on the HPLC instrument used so far. A partial revalidation was done for that on the 7th of January. All acceptance criteria were fulfilled, so the last sample measurement could be performed on the other HPLC instrument. Summary of the method transfer can be seen in Table 4 in 183816 (Appendix 4).

Duration of treatment / exposure:

56 days

Frequency of treatment:

Daily

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

LD (low dose)

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

MD (mid dose)

Dose / conc.:

360 mg/kg bw/day (nominal)

Remarks:

HD (high dose); administration of females with 360 mg/kg bw/day up to premating day 6; females were not dosed on premating days 7 and 8 to allow recovery from their bad health condition on premating day 6 / 7 (dehydration, wasp waist, body weight loss, and diarrhea), administration of males with 360 mg/kg bw/day up to premating day 8

Dose / conc.:

240 mg/kg bw/day (nominal)

Remarks:

HD (high dose); application of males and females with 240 mg/kg bw/day from premating day 9 onwards

No. of animals per sex per dose:

40 females
40 males

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Refer to ‘Supporting, RL1, rat (DRF)/Suqian, 2019/Repeated dose toxicity: oral.001’ study record

Positive control:

None

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 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. The reproductive organs, thyroid/parathyroid glands and all organs showing macroscopic lesions were preserved. The number of implantation sites and corpora lutea was recorded. Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. 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
- Time schedule for examinations: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. All animals were weighed directly before termination. The animal prematurely sacrificed was weighed prior to the sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

HAEMATOLOGY: Yes; haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals (Section 10.20). Blood from the abdominal aorta of the animals was collected in EDTA-coated tubes. The haematological parameters examined are in Table 3. Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals (Section 10.20). Blood from the abdominal aorta of the animals was collected in citrate tubes. The coagulation parameters examined are in Table 4.

CLINICAL CHEMISTRY: Yes; parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals (Section 10.20). Blood from the abdominal aorta of the animals was collected in serum separator tubes. The parameters of clinical biochemistry examined are in Table 5. From all dams and all adult males at termination, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4).

URINALYSIS: Yes; A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/ appearance were recorded. The parameters (Table 6) were measured using qualitative indicators (Henry Schein Urine Stripes URI 10SL).

Oestrous cyclicity (parental animals):

Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity (using vaginal smears). Further on, vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sperm parameters (parental animals):

Parameters examined in P male parental generation: testis weight, epididymis weight

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
Each litter was examined as soon as possible after the delivery of the dam to establish the number and sex of pups, stillbirths, live births, runts and the presence of gross abnormalities. Live pups were counted and sexed and litters weighed within 24 hours of littering (PND 0) and on PND 4 and PND 13. Live pups were identified by tattooing. In addition to the observations of the parent animals, any abnormal behaviour of the offspring were recorded. The anogenital distance (AGD) of each pup was measured on PND 0. Pup body weight measured on PND 0 was converted to cube root and used for the calculation of relative AGD (Relative AGD = AGD/Cube root of pup weight). The number of nipples/areolae in male pups was counted on PND 12.

Hormone assessments: From 2 female pups/litter on day 4 after birth (with the exceptions of female numbers mentioned below) and 2 pups/litter at termination on day 13, blood samples were collected from the defined site in serum separator tubes. All blood samples were stored under appropriate conditions. Blood samples from the day 13 pups were assessed for serum levels for thyroid hormones (T4). On PND 4, pup blood was pooled by litter for thyroid hormone analysis. Two pups per litter were sacrificed on day 4 after birth and blood samples were taken for possible serum hormone assessments. The two pups per litter were female pups to reserve male pups for nipple retention evaluations. No pups were available from female no. 75 which was sacrificed in a moribund condition and female nos. 56 and 59 from the control group and female no. 64 from the MD group which were not pregnant. No pups were eliminated in dam nos. 48 of the control group, no. and 70 of the MD group and nos. 72, 76 and 80 of the HD group as litter size was below 8 pups. As there was only one pup available above a litter size of 8 in dam no. 49 of the control group and dam no. 78 from the HD group, only one pup was sacrificed.

GROSS EXAMINATION OF DEAD PUPS:
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

Postmortem examinations (parental animals):

SACRIFICE
All males were sacrificed any time after the completion of the mating period (after a dosing period of 29 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.

GROSS NECROPSY
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. The ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. The number of corpora lutea and implantation sites was recorded for any females sacrificed on day 26 post-coitum due to non-delivery (female nos. 56 and 59 of the LD group and female no. 64 of the MD group).

HISTOPATHOLOGY / ORGAN WEIGHTS
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals (Table 7).

Tissues from the five randomly selected male and female animals were preserved in 4 % neutral-buffered formaldehyde except for eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they will be transferred to 70 % ethanol. A full histopathology was carried out on the preserved organs and tissues (Table 8) of all animals of the control and high dose groups which were sacrificed at the end of the treatment period. A full histopathology was carried out on the preserved organs and tissues of the animal which was euthanised due to morbidity.

Testes, epididymides, ovaries, uterus with cervix, vagina, accessory sex organs (prostate, seminal vesicle with coagulating gland) were trimmed, embedded into paraffin, cut at an approximated thickness of 2-4 µm and stained with hematoxylin and examined in control and HD animals and in non-pregnant female animals of the LD and MD animals. Testes, epididymides and accessory sex organs (prostate, seminal vesicle with coagulating gland) were also examined in the mating partners of the non-pregnant female LD and MD animals.

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides .

Postmortem examinations (offspring):

SACRIFICE
All surviving pups were killed by cervical dislocation on PND 13. Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.

GROSS NECROPSY
Dead pups and all surviving pups sacrificed on PND 13 were carefully examined externally for gross abnormalities before terminal sacrifice.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid/parathyroid glands from 1 pup/sex/litter/group (if possible) (sacrificed on PND 13) and from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
Thyroid/parathyroid glands from pups were not examined histopathologically as thyroid/parathyroid glands from selected parental males and females showed no toxicologically relevant microscopic findings.

Statistics:

A statistical assessment of the results of litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.

Reproductive indices:

Copulation Index (%) = (No. of rats copulated / No. of pairs) X 100
Fertility Index (%) = (No. of females pregnant / No. of females copulated) X 100
Delivery Index (%) = (No. of dams with live newborns / No. of pregnant dams) X 100

Offspring viability indices:

Viability Index (%) (PND 0-4)
Viability Index (%) (PND 4-13)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 11, Table 12.

Female no. 75, which was euthanized in a moribund condition on premating day 7, was observed with clinical signs of piloerection, hunched posture, and reduced spontaneous activity on the day of sacrifice.

Clinical signs of an impaired health condition of females of the HD group were dehydration (4/10 females), polydipsia/polyuria (9/10 females), diarrhea (1/10 females), hunched posture (7/10 females), prone position (1/10 females), sunken flanks (4/10 females), reduced spontaneous activity (9/10 females), hypothermia (1/10 females) and nasal discharge (1/10 females). These clinical signs occurred in all phases of the treatment period (premating, mating, gestation and lactation) at different grades of severity. On premating day 6 and 7, dehydration, hunched posture and sunken flanks indicated a severely impaired health status in few females of the HD group (female numbers 71-74). As an immediate animal welfare measure of relief treatment of HD females was paused on premating days 7 and 8 and the respective dose level was reduced from 360 mg/kg bw/day to 240 mg/kg bw/day from premating day 9 onwards. Thereafter, the health condition of the females improved. From that time point onwards commonly observed clinical signs of females of the HD group were polydipsia/polyuria and reduced spontaneous activity. 2/10 males of the HD group were observed with diarrhoea on several days of the treatment period.

The clinical sign of reduced spontaneous activity was observed in 1/10 females of the MD group on premating day 7. As it occurred only on one single day of the treatment period it was not considered toxicologically relevant.

The clinical sign of piloerection was intermittently observed in 2/10 females of the control group, 2/10 females of the LD group, 4/10 females of the MD group and 2/10 males and 10/10 females of the HD group. Piloerection could be attributed to a general discomfort of the animals rather than systemic toxicity.
Moving the bedding was observed transiently in 10/10 males and 10/10 females of the MD group as well as 10/10 males and 10/10 females of the HD group. Salivation was noted transiently in 2/10 males of the LD group, 6/10 males and 4/10 females of the MD group, and 10/10 males and 10/10 females of the HD group. Moving the bedding and salivation were seen transiently in timely relation to dose administration and were considered as slight clinical signs elicited by local effects of the test item formulation and/or attributed to discomfort of the animals due to the oral administration, but not systemic toxicity.

Low incidences of slight clinical signs like hairless area in 1/10 females of the control group, 1/10 females of the MD group, 1/10 males and 3/10 females of the HD group were seen without dose dependency and are not considered test item-related. This sign is often observed in animals of this strain and age. The clinical sign of a crust was observed in 1/10 females of the HD group and was assumed incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

For a summary see Table 10.

During the study female no. 75 of the HD group ((360 mg/kg bw/day) was euthanized in moribund condition on premating day 7. The cause of morbidity was related to test item-induced kidney lesions (interstitial fibrosis, tubular dilatation, tubular basophilia, mononuclear and mixed cell infiltrates and tubular degeneration).

All remaining animals (dosed at 240 mg/kg bw/day) survived the scheduled study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 15 to Table 18 and Figure 1 to Figure 5.

Males of the HD group were observed with statistically significantly lower body weight on pre-mating day 7 (p<0.001) and 14 (p<0.001), mating/post-mating day 7 (8% below controls, respectively; p<0.001), mating/post-mating day 15 (p<0.01) and on the day of terminal sacrifice (7% below controls, respectively; p<0.01) when compared to the control group. During the first week of treatment (premating day 1-7; dosed at 360 mg/kg bw/day) males of the HD group on average transiently lost weight. In the further progress of the study (dosed at 240 mg/kg bw/day), males of the HD group gained weight. Thus, as the animals recovered from this transient effect it is not assumed to be toxicologically relevant. Differences in the mean body weight of males of the LD and MD group were within the range of 2% compared to the control group on all days of body weight measurement and were not considered relevant.

Considerable initial but transient statistically significant body weight loss was also observed in female animals of the MD and HD groups during the first week of treatment (premating day 1-7; dosed at 360 mg/kg bw/day; p<0.01). In the further progress of the study (dosed at 240 mg/kg bw/day), all groups gained body weight. In the second week of treatment (premating day 7-14) body weight gain was statistically significantly lower in females of the HD group compared to the control group.

During the later gestation period (gestation day 14 to 20) females of HD group showed a statistically significant lower body weight gain leading to statistically significantly lower body weight on gestation day 20 (9 % below controls; p<0.05). During the lactation period mean body weight was statistically non-significantly below controls (approx. between 7 and 10 %).

The above-mentioned effects on body weights of females of the HD group were considered test item-related.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 19, Table 20, Figure 6 and Figure 7.

In correlation with lower mean body weight of males and females of the HD group, a tendency towards lower food consumption was observed in the HD group during the first and second week of treatment without achieving statistical significance.

Food consumption was statistically significantly lower from lactation day 9-13 (40% below controls).

The above-mentioned effects on food consumption of males and females of the HD group were considered test item-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 31 to Table 34.

The test item had no statistically significant or toxicologically relevant effect on coagulation parameters of male and female animals analyzed in selected animals at the end of the treatment period of this study.

At the end of the treatment period the % of neutrophils in male animals was statistically significant higher in the HD group (p<0.001) when compared to controls (approx. 24 % vs. 14 % in controls). At the same time, the % of lymphocytes was slightly but statistically significant lower in these animals (p<0.001) when compared to controls (approx. 73 % vs. 84 % in controls). In HD females total WBC (p<0.01) and monocytes (p<0.01) were statistically significantly higher than in controls (approx. 67 % and 102 % above controls, respectively). In female animals of the MD or HD groups a tendency towards elevated neutrophils and decreased lymphocytes was observed when compared to controls. The above-mentioned slight changes in white blood cells are possibly related to nephropathy-associated inflammation.

A slight but statistically significant lower MCV level (p<0.01) and higher % of reticulocytes (p<0.01) in male animals of the HD group and a moderate dose-dependent increase in platelets of females of the MD and HD groups (p<0.05, respectively) are not considered toxicologically relevant. Although a test item relation cannot be excluded, the respective values were in the range of historical control data (Appendix 7, Table 1 and Table 2) and thus, changes were not considered adverse.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 35, Table 36.

At the end of the treatment period a tendency towards slightly higher serum urea levels in HD males (approx. 23 % above controls) and statistically significantly higher serum urea (HD group approx. 56 % above controls, p<0.001; MD group approx. 23 % above controls, p<0.05) and crea (HD group approx. 57 % above controls; p<0.001) levels in females were observed. These changes are assumed to be related to nephropathy observed at these dose levels.

Statistically significant increases in serum ALAT level of HD males (deviation from control: 52%; p<0.05) and serum chol of HD females (p<0.01) and slight but statistically significantly decreases in serum ALB in HD females (p<0.01) are not considered toxicologically relevant in absence of other signs of hepatotoxicity and without consistency between genders.

A higher mean TBA level in the females of the HD group compared to control females (189% above controls) was without statistical significance as this was caused by high TBA level of female no. 76 and was rather assumed to be an incidental finding without toxicological relevance.

No test item-related effect of statistical significance or toxicological relevance was observed on adult male thyroxine hormone (T4) levels in the test item-treated groups when compared to the respective controls. For a detailed description of the findings see Table 59.

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

It cannot be excluded that statistically significant effects on estrous cyclicity in the HD group (3/10 females) were caused by treatment with the test item, but there is no historical data available. Also no histological evidence of toxicity was observed in the ovaries of the high dose group, compared to controls. In female pups, slightly but higher, absolute and relative AGD was observed in the HD group when compared controls. Relevant effects in the estrous cyclicity and AGD could indicate the potential of a test item to serve as an endocrine disruptor. However, estrous cyclicity and AGD are not the only parameters to confirm the endocrine disruption and need to be correlated with lot of other parameters like histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid/parathyroid weight and thyroxine hormone (T4). Other parameters showed no findings supporting a possible endocrine disruption modality of the test item. Mean AGD value of females of the HD group was within the range of historical control data, however it cannot be excluded that higher AGD is related to treatment with the test item.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 67 and Table 68.

The test item had no toxicologically relevant effects on urinary parameters analysed in selected animals at the end of the treatment period of this study.

All parameters of the test item-treated groups were not considerably different compared to the corresponding control group and were within the normal range of variation. Isolated findings not considered to be toxicologically relevant were a high amount of leucocytes (500 mg/dl) of 1/5 males (no. 4) of the control group, high amount of glucose (150 mg/dL) in 1/5 males (no. 11) of the LD group, a high amount of protein (500 mg/dL)in 1/5 females (no. 48) of the control group and a high amount of erythrocytes (approx. 250 cells/µL) in the urine of 1/5 females of the MD group (no. 67). As these are isolated findings and occurred only in single animals, this effect was not considered to be test item-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 13, Table 14.

In males and females, no relevant test item related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the controls. There were no biologically relevant differences in body temperature between the groups.

Statistically significantly different body temperature between males of the HD group and control group prior to treatment is not toxicologically relevant. Males of all test item treated groups were observed with statistically significantly higher count of supported rearing compared to the controls (mean count in the control group: 1.40, LD group: 3.20, MD group: 4.40, and HD group: 3.80). Due to the slightness of the difference, the lack of dose-dependency and the absence of effects on other parameters of the functional observation battery these differences were not assumed toxicologically relevant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 13, Table 14

In males and females, no relevant test item related effect was observed in any of the parameters of the functional observation battery at the end of the treatment period when compared to the controls. There were no biologically relevant differences in body temperature between the groups.

Statistically significantly different body temperature between males of the HD group and control group prior to treatment is not toxicologically relevant. Males of all test item treated groups were observed with statistically significantly higher count of supported rearing compared to the controls (mean count in the control group: 1.40, LD group: 3.20, MD group: 4.40, and HD group: 3.80). Due to the slightness of the difference, the lack of dose-dependency and the absence of effects on other parameters of the functional observation battery these differences were not assumed toxicologically relevant.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Reproductive: For a detailed description of the findings see Appendix 3. Under the conditions of this study, the test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

General toxicity: In the kidney of the decedent animal (Animal No. 75) tubular dilatation, tubular basophilia and tubular degeneration were observed. These renal lesions were considered test item related. All other microscopic findings recorded in the decedent animal were within the range of background lesions which may be recorded in animals of this strain and age and in this study type.

In kidneys from high dose group males and females there was a nephropathy characterized by tubular dilatation, tubular basophilia, interstitial fibrosis, mononuclear infiltrates, tubular degeneration, mixed cell infiltrates and transitional cell hyperplasia. In addition, in males and females from the medium dose group nephropathy characterized by tubular dilatation, tubular basophilia and mononuclear infiltrates was also observed in some males and females. When compared between the high and medium dose group the nephropathy observed in the high dose group was of higher incidence and severity than in the medium dose group. Further, no renal changes were observed in animals from the low dose group. The above-mentioned nephropathy observed in several animals from the high and medium dose group was considered test item related.

For a detailed description of the findings see Appendix 3.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 21

The test item had no statistically significant or biologically relevant effect on the estrous cycle analysed during the 2 weeks premating period in females of the LD and MD group when compared to the controls. There were no considerable differences in the length or sequence of cycle stages between the LD and MD group and the control group.

Females of the HD group were observed with statistically significantly lower mean number of normal cycles compared to the control group (1.11 in the HD group compared to 1.90 in the control group). Females of the HD group also showed a tendency towards a higher number of cycles with more than five days (0.22 in the HD group compared to 0.00 in the control group) and consequently a higher mean cycle length (4.57 in the HD group compared to 4.15 in the control group), and a tendency towards a higher number or abnormal cycles (0.22 in the HD group compared to 0.00 in the control group) when compared to control females. Described effects were mainly based on the data of 3/10 females of the HD group which were observed with a persistent diestrous during the premating period; there is no historical control data for oestrous cycle.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

For a detailed description of the findings see Appendix 3. Under the conditions of this study, the test item did not produce any histological evidence of toxicity in the reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

The pre-natal and post-natal parameters including the number of live pups on PND 0, 4 and 13, and pre- and post-implantation loss showed no statistically significant differences between test item-treated groups and the controls. The mean number of corpora lutea (CL) and implantation sites (IS) showed a statistically significantly lower number (24% below controls, respectively; p<0.01) in the HD group and a tendency towards a lower number in the LD and MD groups when compared to the controls. Values are within the range of historical control data (Appendix 6, Table 9), however, a relation to the test item cannot be excluded. For a detailed description of the findings see Table 25

There were no test item-related effects on the reproductive indices including copulation index, fertility index, delivery index (Table 55). Copulation index was 100 % in the control group, 80 % in the LD group, 90 % in the MD group and 100% in the HD group. Fertility and delivery indices were 100 % in all groups. Slight differences in the reproductive indices of test item-treated females compared to control females followed no dose-dependency and were within the normal range of variation. They were not assumed to be toxicologically relevant.

Precoital interval and duration of gestation were not affected by treatment with the test item. The test item groups showed no statistically significant differences in precoital interval and duration of gestation when compared to the control group (For a detailed description of the findings see Table 24 and Table 53).

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The survival of the pups from PND 0 to PND 4 and from PND 4 to PND 13 was not statistically significantly affected in test item groups when compared to the control group.Observed mortality in the control group (1/12 pups from dam no. 41 was found dead on PND 1 and 1/8 pups from dam no. 42 was found dead on PND 13) and the LD group (1/9 pups from dam no. 51, 1/11 pups from dam no. 53, and 1/9 pups from dam no. 55 were found dead on PND 13) was within the normal range of variation and not considered to be toxicologically relevant. No mortality was observed in the MD and HD group from PND 0 to PND 4 and from PND 4 to PND 13. (For a detailed description of the findings see Table 27)

There were no test item-related effects on the viability index (Table 26). Slightly lower viability index (PND 0-4) in the control group (99.17 %) was caused by 1/10 females and was considered as biological variation. Viability index (PND 0-4) was 100 % in the LD, MD, and HD group. Slightly lower viability indices (PND 4-13) in the control (98.75 %) and the LD group (96.09%) were caused by single animals and were considered as biological variation and without toxicological relevance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup weight was shown to be dose-dependently lower on PND 0 (C: 6.33g, LD: 6.09g, MD: 5.93g, HD: 5.63g), PND 4 (C: 11.30g, LD: 10.49, MD: 10.33, HD: 5.63g), and PND 13 (C: 29.91g, LD: 28.61, MD: 26.93, HD: 23.55g) with achieving statistical significance in the HD group compared to the control group on PND 4 (p<0.05) and PND 13 (p<0.01). The values were within the range of historical control data (Appendix 6: Historical Control Data, Table 7), however, as this effect showed dose-dependency and statistical significance in the HD group it was considered to be test item related. For a detailed description of the findings see Table 22

Male litter weight was statistically significantly lower in the HD group compared to the male control pups on PND 0 (33% below control; p<0.05), PND 4 (42% below control; p<0.01), and PND 13 (43% below control; p<0.01). Consequently, total litter weight was statistically significantly lower on PND 0 (27% below control; p<0.05), PND 4 (34% below control; p<0.01), and PND 13 (31% below control; p<0.01) when comparing pups of the HD group with control pups. Values are within the range of historical control data (Appendix 6, Table 7), however, a relation to the test item cannot be excluded. Treatment with the test item had no statistically significant effect on female litter weight data on PND 0, 4 and 13 when comparing test item-treated groups and the controls.
For a detailed description of the findings see Table 23 and Table 52.

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:

no effects observed

Description (incidence and severity):

No test item-related effect of statistical significance or toxicological relevance was observed on male or female PND 13 pup thyroxine hormone (T4) levels in the test item-treated groups when compared to the respective controls. For a detailed description of the findings see Table 30

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 28
Absolute anogenital distance (AGD) of male pups was statistically significantly shorter (p<0.05) in the MD group (2.44mm) when compared to male control pups (2.64mm). However, relative AGD showed no statistically significant differences in male pups of the test item-treated groups compared to the controls.
However, as this parameter not affected in the LD and HD group, the statistically significant difference of the MD group was not considered toxicologically relevant.

In female pups, slightly but statistically significantly longer, absolute and relative AGD was observed in females of the HD group when compared to the female control pups (absolute AGD: 1.20 mm vs. 1.00 mm in controls; p<0.01 and relative AGD: 0.68 vs. 0.55 in controls; p<0.001). Values are within the range of historical control data (Appendix 6, Table 27), however, a relation to the test item cannot be excluded.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 28

Mean nipple retention on PND 12 was observed to be statistically significantly higher (p<0.01) in male pups of the MD group (0.94) compared to male pups of the control group (0.35). However, as this parameter was not affected in the LD and HD group, the statistically significant difference of the MD group was not considered toxicologically relevant.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean thyroid/parathyroid weight of male and female pups of the test item-treated groups was comparable to mean thyroid/parathyroid weight of corresponding control pups. Differences between the groups were within the normal range of variation and were not caused by treatment with the test item (Table 58).

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related gross external abnormalities of toxicological relevance were observed in the pups of any of the groups on PND 0-12. Few findings in the control group like a dark and small pup (1/12 pups from dam no. 41, PND 0), in the LD group like a small pup (1/11 pups from dam no. 55, PND 0) and a pup with a dark snout (1/11 pups from dam no. 55, PND 0), in the MD group like one small pup (1/11 pups from dam no. 67), and in the HD group like hairless area on the back (9/11 pups from dam no. 77) were observed without dose-dependency. These findings are considered to be incidental and not related to treatment with the test item.

The external findings of hairless area on the back in the control group (12/14 pups from dam no. 43), hairless area on back and neck in the MD group (6/6 pups from dam no. 70), hairless area on back and neck in the HD group (7/7 pups from dam no. 72), and dehydration, underweight, and no indication of suckling in the LD group (1/11 pups from dam no. 55) at death were considered to be spontaneous and not related to test item treatment.

For a detailed description of the findings see Table 69.

Histopathological findings:

not examined

Description (incidence and severity):

Thyroid/parathyroid glands from pups were not examined as thyroid/parathyroid glands from selected parental males and females showed no toxicologically relevant microscopic findings.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

No statistically significant test item effects were observed in any of the dose groups on litter data parameters like total number of pups born (alive and dead), number of male pups, number of female pups, sex ratio, number of live pups, still birth, runt on PND 0 as well as number of live pups, number of male pups, number of female pups and sex ratio on PND 4 and PND 13 when compared with the controls.

However, a slight tendency towards a lower mean total number of pups (alive and dead) was observed in the HD group compared to control females on PND 0 (8.89 in HD group compared to 10.60 in the control group) but it was not significant. (For a detailed description of the findings see Table 22)

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

120 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

sexual maturation

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

no

Lowest effective dose / conc.:

120 mg/kg bw/day (nominal)

Treatment related:

no

Conclusions:

Based on the findings of this combined repeated dose and reproduction/developmental toxicity screening test in Wistar rats, the NOAEL (parental/offspring) of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine for for reproductive toxicity screening is considered to be 120 mg/kg bw/day.

Executive summary:

In a combined repeated dose and reproduction/developmental toxicity screening test (183812), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) was administered to 4 groups of Wistar rats (10 animals/sex/group) by gavage in corn oil at dose levels of 0, 40 (LD), 120 (MD), and 360/240 (HD) mg/kg body weight/day, 7 days per week. Females were administered 360 mg/kg bw/day up to premating day 6; females were not dosed on premating days 7 and 8 to allow recovery from their bad health condition on premating day 6/7. Males were administered 360 mg/kg bw/day up to premating day 6. Males and females were administered 240 mg/kg bw/day from premating day 9 onwards. The animals were treated for a maximum period of 56 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

 

Concentration analysis and homogeneity of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 90/60 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The concentration of the HD dose group was reduced in week 3. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %. All samples were homogenous, as COV was below or equal 10%.

 

Treatment with the test item caused mortality in 1/10 females of the HD group (360 mg/kg bw/day). Morbidity in this female was caused by kidney lesions (tubular dilatation, tubular basophilia and tubular degeneration) caused by treatment with the test item. All remaining animals (dosed at 240 mg/kg bw/day) survived the scheduled study period.

 

Clinical symptoms indicating systemic toxicity caused by test item were observed in the HD group. Due to increasingly poor health condition dosing of HD females their dosing was stopped on premating days 7 and 8 and dosing of males and females of the HD group was continued a with a reduced dose level of 240 mg/kg/day from premating day 9 onwards what led to improvement of the health condition especially in females. No clinical signs of systemic toxicity were observed at the MD and LD levels.

 

Treatment with the test item temporarily affected body weights of male animals of the HD group and female animals of the MD and HD groups. No effect was observed at the LD level. In correlation with effects of the test item on body weights of the HD group, a tendency towards lower food consumption was observed in male and female animals of the HD group during the first and second week of treatment. In females of the HD group statistically significantly lower food consumption was noted towards the end of the lactation period.

 

Higher total WBC and monocytes in HD females compared to controls observed at the end of the treatment period are possibly related to nephropathy-associated inflammation. No toxicologically relevant effect of the test item was observed at LD and MD levels. Statistically significant effects in MCV level and % of reticulocytes in males (HD) and platelets in females (MD and HD) were not considered adverse as values were within the range of historical control data. However, it cannot be excluded that mentioned differences were caused by treatment with the test item. At the end of the treatment period a tendency towards slightly higher serum urea levels in HD males and higher serum urea levels (HD and MD group) and crea levels (HD group) were observed in female animals which were assumed to be related to nephropathy observed at the corresponding dose level. The test item had no toxicologically relevant effects on urinary parameters analysed at the end of the treatment period of this study.

 

Test item-related necropsy findings in the HD group consisted of enlarged kidneys (5/10 males and 8/10 females) and kidneys which were observed with an abnormal color (5/10 males), shape (1/10 females), or surface (observed in 8/10 females and 1/10 males) and correlated with histopathology lesions. No other test item-related macroscopic findings were noted in any of the groups. Marked and statistically significant higher kidney weight in females of the HD group correlates with the histopathology changes and was considered test item-related. Higher kidney weight was seen in males of the HD group. All other organ weight changes were considered of no toxicological relevance due to the absence of correlating histological lesions. Test item-related kidney nephropathy was observed in the MD and HD group. In kidneys of HD males and females there was nephropathy characterized by tubular dilatation, tubular basophilia, interstitial fibrosis, mononuclear infiltrates, tubular degeneration, mixed cell infiltrates and transitional cell hyperplasia. Nephropathy characterized by tubular dilatation, tubular basophilia and mononuclear infiltrates were observed in the kidneys of some males and females of the MD group. No renal changes were observed in animals from the LD group.

 

Under the conditions of this study, no histological evidence of toxicity was observed in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

 

No statistically significant test item effects were observed on litter data parameters. Mean number of corpora lutea (CL) and implantation sites (IS) showed statistically significantly lower in the HD group and LD and MD groups showed a tendency towards lower CL and IS numbers. Although statistically significant, lower CL and IS values in the HD group were within the range of historical control data. However, an effect of the test item on CL and IS cannot be excluded.

 

Mean male litter weight and consequently total litter weight were statistically significantly lower in the HD group compared to the male control pups on PND 0, PND 4, and PND 13. It cannot be excluded that this condition is caused by treatment of parental animals with the test item. Though, value of lower male litter weight was within the range of historical control data. Dose-dependently lower mean pup weight on PND 0, PND 4, and PND 13 achieving statistical significance in the HD group on PND 4 and PND 13 and lower mean male and female pup weight in test item-treated groups on PND 0 resulting in significantly lower mean weight of male and female pups of the HD group were considered test item related based on the dose-dependency and statistical significance in the HD group. Mean pup weight and litter weight were not affected in the MD and LD groups.

 

It cannot be excluded that statistically significant effects on estrous cyclicity in the HD group (3/10 females) were caused by treatment with the test item, but there is no historical data available. Also no histological evidence of toxicity was observed in the ovaries of the high dose group, compared to controls. In female pups, slightly but higher, absolute and relative AGD was observed in the HD group when compared controls. Relevant effects in the estrous cyclicity and AGD could indicate the potential of a test item to serve as an endocrine disruptor. However, estrous cyclicity and AGD are not the only parameters to confirm the endocrine disruption and need to be correlated with lot of other parameters like histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid/parathyroid weight and thyroxine hormone (T4). Other parameters showed no findings supporting a possible endocrine disruption modality of the test item. Mean AGD value of females of the HD group was within the range of historical control data, however it cannot be excluded that higher AGD is related to treatment with the test item.

 

Based on the findings of this study the NOAEL (parental/offspring) of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine for reproductive toxicity screening is considered to be 120 mg/kg bw/day.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

Results from the OECD 422/GLP study (RL1) shows no adverse effects on reproductive organs or tissues in males or females, so the extended one-generation reproductive toxicity study (OECD 443) is waived.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was an OECD 422/GLP study and is the only study available. It was assigned a Klimisch score of 1.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to Reproduction (Screening):

There is one dose range finding (DRF) study in rats available and one Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in rats available.

 

 

Dose range finding (DRF) study

In a dose range finding study for a reproduction/developmental toxicity screening test (183813), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) was administered to 5 groups of Wistar rats (3 animals/sex/group) by gavage in corn oil at dose levels of 0, 100 (LD), 300 (MD), and 1000 (HD) or 600 (HD) mg/kg body weight/day and additionally 500 (HID) mg/kg body weight/day, 7 days per week. The animals were treated for a period of 54 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

 

Test item-related mortality occurred in 3/3 males and 3/3 females of the HD group. Subsequently, the animals were not treated with the test item on study day 3 and the high dose was reduced from 1000 mg/kg body weight/day to 600 mg/kg body weight/day from study day 4 onwards. Males of the additional group dosed with 500 mg/kg body weight/day were all euthanized in a moribund condition between treatment day 8 and 10.

 

Severe clinical findings in the HD group caused by treatment with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine were reduced spontaneous activity, diarrhea, dehydration, stilted gait and sunken flanks.  Gross pathological changes which may be caused by treatment with the test item were an abnormal colored stomach in one animal of the HD group and masses in the stomach of another HD animal.  No mortality or clinical symptoms indicating systemic toxicity were observed at dose levels of 100 and 300 mg/kg body weight/day.  At a dose level of 300 mg/kg body weight/day (MD) a slightly attenuated body weight gain was observed in males and females.

 

Under the conditions of the study performed, it is assumed that 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine has no toxic effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition as well as pup-related parameters.

 

Based on the data generated from this dose range finding study, dose levels of 40, 120 and 360 mg/kg bw per day are suggested for the subsequent main Combined Repeated Dose Toxicity Study with Reproductive/Developmental Toxicity Screening Test (OECD 422) with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine.

 

Combined Repeated Dose Oral Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

There is one combined repeated dose and reproduction/developmental toxicity screening test in rats available.

 

In a combined repeated dose and reproduction/developmental toxicity screening test (183812), 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (99.29%) was administered to 4 groups of Wistar rats (10 animals/sex/group) by gavage in corn oil at dose levels of 0, 40 (LD), 120 (MD), and 360/240 (HD) mg/kg body weight/day, 7 days per week. Females were administered 360 mg/kg bw/day up to premating day 6; females were not dosed on premating days 7 and 8 to allow recovery from their bad health condition on premating day 6/7. Males were administered 360 mg/kg bw/day up to premating day 6. Males and females were administered 240 mg/kg bw/day from premating day 9 onwards. The animals were treated for a maximum period of 56 days in females, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

 

Concentration analysis and homogeneity of formulation samples was determined at three concentrations, 10 mg/mL, 30 mg/mL and 90/60 mg/mL in study weeks 1, 3, 5 and in the last week of the study. The concentration of the HD dose group was reduced in week 3. Nominal concentrations were confirmed for all dose groups, as measured concentrations were within acceptance criterion of 10 %. All samples were homogenous, as COV was below or equal 10%.

 

Treatment with the test item caused mortality in 1/10 females of the HD group (360 mg/kg bw/day). Morbidity in this female was caused by kidney lesions (tubular dilatation, tubular basophilia and tubular degeneration) caused by treatment with the test item. All remaining animals (dosed at 240 mg/kg bw/day) survived the scheduled study period.

 

Clinical symptoms indicating systemic toxicity caused by test item were observed in the HD group. Due to increasingly poor health condition dosing of HD females their dosing was stopped on premating days 7 and 8 and dosing of males and females of the HD group was continued a with a reduced dose level of 240 mg/kg/day from premating day 9 onwards what led to improvement of the health condition especially in females. No clinical signs of systemic toxicity were observed at the MD and LD levels.

 

Treatment with the test item temporarily affected body weights of male animals of the HD group and female animals of the MD and HD groups. No effect was observed at the LD level. In correlation with effects of the test item on body weights of the HD group, a tendency towards lower food consumption was observed in male and female animals of the HD group during the first and second week of treatment. In females of the HD group statistically significantly lower food consumption was noted towards the end of the lactation period.

 

Higher total WBC and monocytes in HD females compared to controls observed at the end of the treatment period are possibly related to nephropathy-associated inflammation. No toxicologically relevant effect of the test item was observed at LD and MD levels. Statistically significant effects in MCV level and % of reticulocytes in males (HD) and platelets in females (MD and HD) were not considered adverse as values were within the range of historical control data. However, it cannot be excluded that mentioned differences were caused by treatment with the test item. At the end of the treatment period a tendency towards slightly higher serum urea levels in HD males and higher serum urea levels (HD and MD group) and crea levels (HD group) were observed in female animals which were assumed to be related to nephropathy observed at the corresponding dose level. The test item had no toxicologically relevant effects on urinary parameters analysed at the end of the treatment period of this study.

 

Test item-related necropsy findings in the HD group consisted of enlarged kidneys (5/10 males and 8/10 females) and kidneys which were observed with an abnormal color (5/10 males), shape (1/10 females), or surface (observed in 8/10 females and 1/10 males) and correlated with histopathology lesions. No other test item-related macroscopic findings were noted in any of the groups. Marked and statistically significant higher kidney weight in females of the HD group correlates with the histopathology changes and was considered test item-related. Higher kidney weight was seen in males of the HD group. All other organ weight changes were considered of no toxicological relevance due to the absence of correlating histological lesions. Test item-related kidney nephropathy was observed in the MD and HD group. In kidneys of HD males and females there was nephropathy characterized by tubular dilatation, tubular basophilia, interstitial fibrosis, mononuclear infiltrates, tubular degeneration, mixed cell infiltrates and transitional cell hyperplasia. Nephropathy characterized by tubular dilatation, tubular basophilia and mononuclear infiltrates were observed in the kidneys of some males and females of the MD group. No renal changes were observed in animals from the LD group.

 

Under the conditions of this study, no histological evidence of toxicity was observed in reproductive organs and tissues including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus and cervix, and vagina. Further, there were no treatment-related effects on the testicular histomorphology including spermatogenesis and interstitial cell structure.

 

No statistically significant test item effects were observed on litter data parameters. Mean number of corpora lutea (CL) and implantation sites (IS) showed statistically significantly lower in the HD group and LD and MD groups showed a tendency towards lower CL and IS numbers. Although statistically significant, lower CL and IS values in the HD group were within the range of historical control data. However, an effect of the test item on CL and IS cannot be excluded.

 

Mean male litter weight and consequently total litter weight were statistically significantly lower in the HD group compared to the male control pups on PND 0, PND 4, and PND 13. It cannot be excluded that this condition is caused by treatment of parental animals with the test item. Though, value of lower male litter weight was within the range of historical control data. Dose-dependently lower mean pup weight on PND 0, PND 4, and PND 13 achieving statistical significance in the HD group on PND 4 and PND 13 and lower mean male and female pup weight in test item-treated groups on PND 0 resulting in significantly lower mean weight of male and female pups of the HD group were considered test item related based on the dose-dependency and statistical significance in the HD group. Mean pup weight and litter weight were not affected in the MD and LD groups.

 

It cannot be excluded that statistically significant effects on estrous cyclicity in the HD group (3/10 females) were caused by treatment with the test item, but there is no historical data available. Also no histological evidence of toxicity was observed in the ovaries of the high dose group, compared to controls. In female pups, slightly but higher, absolute and relative AGD was observed in the HD group when compared controls. Relevant effects in the estrous cyclicity and AGD could indicate the potential of a test item to serve as an endocrine disruptor. However, estrous cyclicity and AGD are not the only parameters to confirm the endocrine disruption and need to be correlated with lot of other parameters like histopathology of parental reproductive organs and their weights, litter size, sex ratio, pup thyroid/parathyroid weight and thyroxine hormone (T4). Other parameters showed no findings supporting a possible endocrine disruption modality of the test item. Mean AGD value of females of the HD group was within the range of historical control data, however it cannot be excluded that higher AGD is related to treatment with the test item.

 

Based on the findings of this study the NOAEL (parental/offspring) of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine for reproductive toxicity screening is considered to be 120 mg/kg bw/day.

 

Extended one-generation reproductive toxicity:

Results from the OECD 422/GLP and OECD 414/GLP studies (RL1) shows no adverse effects on reproductive organs or tissues in males or females, so the extended one-generation reproductive toxicity study (OECD 443) is waived.

Effects on developmental toxicity

Description of key information

Pre-natal developmental toxicity in rats

NOAEL (maternal/foetotoxicity) = 50 mg/kg bw/day (OECD 414/GLP)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Remarks:

DRF for OECD 414 study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 March 2022 - 24 October 2022

Reliability:

1 (reliable without restriction)

Qualifier:

no guideline required

Principles of method if other than guideline:

This study was designed as a dose range finding study and in this respect followed the procedures as indicated in the following internationally accepted guidelines and recommendation: OECD 414

GLP compliance:

no

Remarks:

DRF study, GLP not required

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

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: 10-11 weeks old

- Weight at study initiation: males: 354 – 438 g (mean: 418 g, ± 20% = 335.02 – 502.53 g); females: 198 – 253 g (mean: 216.29 g, ± 20% = 173.04 – 259.55 g)

- Housing: The animals were kept 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 (e.g. ad libitum): Free access to Altromin 1324 maintenance diet for rats and mice

- Water (e.g. ad libitum): 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

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The formulation was homogenized by subjecting it to a water bath (40°C) until visual homogeneity was achieved (at least 30 min). Formulates was kept under magnetic stirring during the daily administration.


VEHICLE
The test item was dissolved/suspended in corn oil which was used in previous in vivo studies.

Analytical verification of doses or concentrations:

no

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 evidence of mating.
The day on which sperm is 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 32 sperm-positive females, the remaining females and males were discarded
without any observation. However, in case of an increased number of non-pregnant females in any of
the groups, the remaining females were mated and assigned to the control and/or treated groups in
order to obtain about 7 pregnant females per group.

Duration of treatment / exposure:

GD 5 to GD 19

Frequency of treatment:

Daily

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Control group - 7 females
Dose groups - 8 females

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the previously conducted OECD 422 test (Eurofins study number 183812), the doses selected were 0, 50, 150 and 300 mg/kg bw/day

Maternal examinations:

CAGE SIDE OBSERVATIONS: Prior to the start of the mating a detailed clinical observation outside the home cage was made

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

FOOD CONSUMPTION: Food consumption of sperm-positive females was determined for the following intervals: GDs 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: On GD 20, sperm-positive (presumed pregnant) females were subjected to a caesarean section after sacrificing the animals using an overdose of sodium pentobarbital. 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: 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.

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. Lip and palate were examined for cleft lip and palate by gently opening the mouth with forceps. The head, eyes, ears, jaw and snout were 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.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A single incidence of abnormal breathing and regurgitation (at post dose) was observed in LD group (animal no. 13) on GD 12 and moderate salivation in LD animal no. 15 on GD19. Moving the bedding (2/8 in LD group, 3/8 in MD group and 8/8 in HD group) and increased salivation, slight to severe (1/8 in LD group, 5/8 in MD group and 4/8 in HD group) during different days of gestation were observed. Moving the bedding or increased salivation were considered to be a signs of local reaction to the test item. Polydipsia and diarrhoea were recorded in 1/8 HD group on GD 20. Hairless area in 1/7 control and 1/8 in HD group, scratch/cut in 1/8 HD group were noticed. All these signs were considered to be incidental findings.

Mortality:

mortality observed, treatment-related

Description (incidence):

HD group animal no. 29 was moribund sacrificed on GD 18 for the animal welfare reasons. It showed clinical signs of reduced spontaneous activity, abnormal breathing, piloerection, hunched posture, half eye closure, wasp waist, regurgitation, nasal discharge and dehydration from GD 9-11 and GD 16-18. Body weight was slightly reduced on GD 14-17 (9.25%) and lowered food consumption. At necropsy, it had fluid filled thoracic cavity (red, mucoid), lungs with white fluid filled and failure to collapse. The possible cause of moribund condition could be regurgitation of test item formulation at post dose from GD9 and this finding can't be excluded from the test item related effects. All other rats survived until the end of the study (GD20).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At day 20, the mean body weights of treated groups were found to be comparable to the control group and there were no statistically significant changes observed. Animals in the LD group gained weight slightly (5.75%) while animals in the MD and HD lost weight slightly (1.19% and 5.14%, respectively), compared to the control group. The mean body weight gain was decreased in MD group on GDs 5-8 (76.52%), GDs 11-14 (12.50%) and on GDs 17-20 (26.47%) when compared to control. Statistically significant decrease in mean body weight gain was observed in HD group on GDs 5-8 (154.07%; p<0.05), GDs 8-11 (83.15%; p<0.01) and on GDs 17-20 (70.97%; p<0.05) when compared to control. Overall, on GD 5-20 there were reduction in mean body weight gain in MD (14.89%) and HD (41.77%; p<0.01) groups observed when compared to control. These mean body weight reductions were considered to be test item-related effects.

Slight but statistically significant reduction in mean carcass weight was observed in HD group (11.64%; p<0.05) when compared to control. It is considered to be a test item-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In correlation to mean body weight gain effects in the HD group, mean food consumption was found to be reduced on GDs 5-8 (22.58%), GDs 8-11 (22.33%), GDs 11-14 (14.67%), GDs 14-17 (11.99%) and GDs 17-20 (38.43%; p<0.05) in HD groups when compared to control. In the MD group, there was a slight reduction in mean food consumption observed on GDs 8-11 (13.72%) and on GD 17-20 (15.44%). The mean food consumption of LD group was comparable to controls on GDs 5-20. Overall, the mean food consumption was affected by treatment with the test item in MD and HD groups on GDs 5-20 (12.39% and 22.50% % (p<0.05) respectively, below control).

Food efficiency:

not examined

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

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test item-related effects were observed for uterine weight in the treatment groups when compared to the control group

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination revealed spotted appearance of kidneys in 1/8 females of MD group and 2/8 females of HD group. Enlarged adrenal glands was noticed in 1/8 females of MD group and 1/8 females of HD group. Red coloured fundic area in stomach with clear mucoid content, spotted appearance of liver in all lobes and small spleen were observed in 1/8 females of HD group. All these findings are considered to be incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Number of abortions:

no effects observed

Description (incidence and severity):

None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No test item-related effects were observed for pre- and post-implantation loss in the treatment groups when compared to the control group

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

No test item-related effects were observed for early and late resorptions in the treatment groups when compared to the control group

Early or late resorptions:

no effects observed

Description (incidence and severity):

No test item-related effects were observed for early and late resorptions in the treatment groups when compared to the control group

Dead fetuses:

no effects observed

Description (incidence and severity):

No dead foetuses were noted in any of the groups.

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Mating resulted in 8/8, 8/8 and 7/8 pregnancies in the LD, MD and HD groups respectively and 7/7 pregnancies in the control group.

Other effects:

no effects observed

Description (incidence and severity):

No test item-related effects were observed for number of corpora lutea and implantation sites in the treatment groups when compared to the control group

Dose descriptor:

other: No NOAEL; doses selected for main study

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight but statistically significant lowered mean fetal body weight was observed in the MD and HD groups (Male: 7.88% and 12.20% respectively; p<0.001, and Female: 8.22% and 14.14% respectively; p<0.001, below control). The change in foetal weight in MD and HD groups were considered to be test item-related and secondary effects of maternal toxicity (lower body weight and food consumption).

Reduction in number of live offspring:

not examined

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The test item had no effect on the sex ratio of the foetuses per dam.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Slight but statistically significant reduction in mean litter weights was observed in HD group (p<0.05) and HD group fetus weight of female fetuses (p<0.05).

Anogenital distance of all rodent fetuses:

not examined

External malformations:

no effects observed

Description (incidence and severity):

No test item related external abnormalities were observed in the foetuses of treated groups.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The number of foetuses per dam was comparable in the treated groups when compared to the control.

Dose descriptor:

other: No NOAEL; doses selected for main study

Developmental effects observed:

no

Conclusions:

Based on the data generated from this dose range finding study, dose levels of 50, 150, 275 mg/kg bw/day are suggested for the subsequent main study with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine.

Executive summary:

In a dose range finding study (no guideline) for a prenatal developmental toxicity in rats (OECD 414/GLP), the test item (99.29%) was administered to pregnant Wistar, Crl: WI(Han) rats (8/dose) by oral gavage in corn oil at dose levels of 0 (7 animals), 50, 150 and 300 mg/kg bw/day daily from GD 5-19.

One HD group (animal no. 29) was moribund sacrificed on GD 18 for the animal welfare reasons. It showed clinical signs of reduced spontaneous activity, abnormal breathing, piloerection, hunched posture, half eye closure, wasp waist, regurgitation, nasal discharge and dehydration from GD 9-11 and GD 16-18. Body weight was slightly reduced on GD 14-17 (9.25%) and lowered food consumption. At necropsy, it had fluid filled thoracic cavity (red, mucoid), lungs with white fluid filled and failure to collapse. The possible cause of moribund condition could be regurgitation of test item at post dose from GD9. All remaining females survived until their scheduled sacrifice.

On GD 20, the mean body weights of treated groups were found to be comparable to the control group and there were no statistically significant changes observed. Animals in the LD group gained weight slightly (5.75%) while animals in the MD and HD lost weight slightly (1.19% and 5.14%, respectively), compared to the control group; however, the mean body weight gain was affected in MD (14.89%) and HD (41.77%; p<0.01) groups on GDs 5-20 when compared to control and the reduction was considered to be test item-related.

In correlation to mean body weight gain effects in the HD group, mean food consumption was found to be reduced on GDs 5-8 (22.58%), GDs 8-11 (22.33%), GDs 11-14 (14.67%), GDs 14-17 (11.99%) and GDs 17-20 (38.43%; p<0.05) in HD groups when compared to control. In the MD group, there was a slight reduction in mean food consumption observed on GDs 8-11 (13.72%) and on GD 17-20 (15.44%). The mean food consumption of LD group was comparable to controls on GDs 5-20. Overall, the mean food consumption was affected by treatment with the test item in MD and HD groups on GDs 5-20 (12.39% and 22.50% (p<0.05) respectively, below control).

 

Mating resulted in 8/8, 8/8 and 7/8 pregnancies in the LD, MD and HD groups respectively and 7/7 pregnancies in the control group. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice. Slight but statistically significant reduction in mean carcass weight was observed in HD group (11.64%; p<0.05) when compared to control. It is considered to be a test item-related effect. No test item-related effects were observed for other prenatal parameters including uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre- and post-implantation loss in the treatment groups when compared to the control group. No dead foetuses were noted in any of the groups. In addition, there were no considerable differences in prenatal parameters between the treated groups and the control group.

 

The number of foetuses per dam was comparable in the treated groups when compared to the control. The test item had no effect on the sex ratio of the foetuses per dam. Slight but statistically significant reduction in mean litter weights was observed in HD group (p<0.05) and HD group fetus weight of female fetuses (p<0.05). Slight but statistically significant lowered mean fetal body weight was observed in the MD and HD groups (Male: 7.88% and 12.20% respectively; p<0.001 and Female: 8.22% and 14.14% respectively; p<0.001, below control). The change in foetal weight in MD and HD groups were considered to be test item-related and secondary effects of maternal toxicity (lower body weight and food consumption). No test item- related effects were observed in the LD group when compared to control. No test item related external abnormalities were observed in the foetuses of treated groups. No test item-related macroscopic lesions were observed in the treated groups.

 

Based on the data generated from this dose range finding study, dose levels of 50, 150, 275 mg/kg bw/day are suggested for the subsequent main study with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)- 1,3,5-triazin-2-amine.

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2018

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Low Dose (LD)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Mid Dose (MD)

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

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).

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

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

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

gross pathology

Abnormalities:

no effects observed

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.

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

Abnormalities:

no effects observed

Developmental effects observed:

no

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.

 

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study was an OECD 414/GLP study and is the only study available. It was assigned a Klimisch score of 1.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Pre-natal developmental toxicity
There is one pre-natal developmental toxicity study in the rat available.

Dose range-finding study for prenatal developmental toxicity study in rats.

In a dose range finding study (no guideline) for a prenatal developmental toxicity in rats (OECD 414/GLP), the test item (99.29%) was administered to pregnant Wistar, Crl: WI(Han) rats (8/dose) by oral gavage in corn oil at dose levels of 0 (7 animals), 50, 150 and 300 mg/kg bw/day daily from GD 5-19.

One HD group (animal no. 29) was moribund sacrificed on GD 18 for the animal welfare reasons. It showed clinical signs of reduced spontaneous activity, abnormal breathing, piloerection, hunched posture, half eye closure, wasp waist, regurgitation, nasal discharge and dehydration from GD 9-11 and GD 16-18. Body weight was slightly reduced on GD 14-17 (9.25%) and lowered food consumption. At necropsy, it had fluid filled thoracic cavity (red, mucoid), lungs with white fluid filled and failure to collapse. The possible cause of moribund condition could be regurgitation of test item at post dose from GD9. All remaining females survived until their scheduled sacrifice.

On GD 20, the mean body weights of treated groups were found to be comparable to the control group and there were no statistically significant changes observed. Animals in the LD group gained weight slightly (5.75%) while animals in the MD and HD lost weight slightly (1.19% and 5.14%, respectively), compared to the control group; however, the mean body weight gain was affected in MD (14.89%) and HD (41.77%; p<0.01) groups on GDs 5-20 when compared to control and the reduction was considered to be test item-related.

In correlation to mean body weight gain effects in the HD group, mean food consumption was found to be reduced on GDs 5-8 (22.58%), GDs 8-11 (22.33%), GDs 11-14 (14.67%), GDs 14-17 (11.99%) and GDs 17-20 (38.43%; p<0.05) in HD groups when compared to control. In the MD group, there was a slight reduction in mean food consumption observed on GDs 8-11 (13.72%) and on GD 17-20 (15.44%). The mean food consumption of LD group was comparable to controls on GDs 5-20. Overall, the mean food consumption was affected by treatment with the test item in MD and HD groups on GDs 5-20 (12.39% and 22.50% (p<0.05) respectively, below control).

 

Mating resulted in 8/8, 8/8 and 7/8 pregnancies in the LD, MD and HD groups respectively and 7/7 pregnancies in the control group. None of the females showed signs of abortion or premature delivery prior to the scheduled sacrifice. Slight but statistically significant reduction in mean carcass weight was observed in HD group (11.64%; p<0.05) when compared to control. It is considered to be a test item-related effect. No test item-related effects were observed for other prenatal parameters including uterine weight, number of corpora lutea, implantation sites, early and late resorptions, pre- and post-implantation loss in the treatment groups when compared to the control group. No dead foetuses were noted in any of the groups. In addition, there were no considerable differences in prenatal parameters between the treated groups and the control group.

 

The number of foetuses per dam was comparable in the treated groups when compared to the control. The test item had no effect on the sex ratio of the foetuses per dam. Slight but statistically significant reduction in mean litter weights was observed in HD group (p<0.05) and HD group fetus weight of female fetuses (p<0.05). Slight but statistically significant lowered mean fetal body weight was observed in the MD and HD groups (Male: 7.88% and 12.20% respectively; p<0.001 and Female: 8.22% and 14.14% respectively; p<0.001, below control). The change in foetal weight in MD and HD groups were considered to be test item-related and secondary effects of maternal toxicity (lower body weight and food consumption). No test item- related effects were observed in the LD group when compared to control. No test item related external abnormalities were observed in the foetuses of treated groups. No test item-related macroscopic lesions were observed in the treated groups.

 

Based on the data generated from this dose range finding study, dose levels of 50, 150, 275 mg/kg bw/day are suggested for the subsequent main study with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)- 1,3,5-triazin-2-amine.

 

Prenatal developmental toxicity study in rats

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.

Justification for classification or non-classification

Based on available information in the dossier, the substance 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine (CAS No. 72058-41-4) does not need to be classified for reproductive toxicity when considering the criteria outlined in Annex I of 1272/2008/EC.