Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

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

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

Link to relevant study records
Reference
Endpoint:
reproductive toxicity, other
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 -
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
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 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:
63 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, Table 40 and Table 41.

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, Table 44 to Table 47 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, Table 48, Table 49, 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, non-treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 31 to Table 34 and Table 61 to Table 64
.
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 (see Section 12.21).

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 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, Table 65 and Table 66.

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 (see Section 12.21).

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.

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, Table 42 and Table 43.

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):
Reproductive: Absolute mean prostate weight (with seminal vesicles and coagulating glands) was statistically significant lower in males of the HD group compared to males of the control group (deviation from control 12 %, p<0.01). Differences in mean organ weights of test item-treated males compared to control males showed no dose-dependency. For a detailed description of the findings see Table 38 and Table 71.

General toxicity: Markedly and statistically significantly higher absolute (p<0.001) and relative (p<0.001) mean kidney weight was observed in female animals of the HD group when compared to controls (deviation from control group: 59 % and 76 %, respectively). Higher mean kidney weight correlates with the histopathology changes and was considered test item-related. Also, males of the HD group showed slightly higher relative mean kidney weight when compared to control males (deviation from control: 19 %);. hHowever, this difference was not statistically significant. Besides statistically significant higher mean kidney weight of HD females compared to control females, all other organ weight changes were considered of no toxicological relevance due to the absence of correlating histological lesions. For a detailed description of the findings see Table 38 and Table 71.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 37

Reproductive: The isolated finding of small prostate gland and seminal vesicles in 1/10 males of the HD group was considered incidental without the correlation of histological lesions and not assumed test item-related. Besides these findings, no other macroscopic findings were noted in animals of the HD group or at the LD and MD levels.

General toxicity: Test item related necropsy findings correlating with histopathology lesions were observed in kidneys of several males and females from the HD group. These findings 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). Besides these findings, no other macroscopic findings were noted in animals of the HD group or at the LD and MD levels.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 13, Table 14, Table 42 and Table 43.

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 (Table 9). The above-mentioned nephropathy observed in several animals from the high and medium dose group was considered test item related.
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 21 and Table 50.

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. It cannot be excluded that persistent diestrous was caused by treatment with the test item.
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) in the HD group and a tendency towards a lower number in the LD and MD groups when compared to the controls. For a detailed description of the findings see Table 25 and Table 54.

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 function (oestrous cycle)
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 and Table 56.)

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):
For a detailed description of the findings see Table 23 and Table 52.

Male litter weight was statistically significantly lower in the HD group compared to the male control pups on PND 0 (33% below control), PND 4 (42% below control), and PND 13 (43% below control). Consequently, total litter weight was statistically significantly lower on PND 0 (27% below control), PND 4 (34% below control), and PND 13 (31% below control) when comparing pups of the HD group with control pups.

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.
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 and Table 60.
Urinalysis findings:
not examined
Sexual maturation:
not examined
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, 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). (For a detailed description of the findings see Table 22 and Table 51.)
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
mortality
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

Table 9: Incidence and mean severity of renal findings in surviving animals

Dose (mg/kg bw/day)

0

40

120

360*/240**

Animals per Sex /

Affected / Mean Grade

5 M

5 F

5 M

5 F

5M

5 F

10 M

9 F

Tubular dilatation

-

-

-

-

1/1.0

1/3.0

9/3.1

9/3.9

Tubular basophilia

-

-

-

-

2/1.0

4/2.0

9/2.6

9/3.1

Interstitial fibrosis

-

-

-

-

1/2.0

1/2.0

9/2.2

9/3.4

Mononuclear infiltrates

-

-

-

-

2/1.5

4/1.8

9/2.3

9/3.2

Tubular degeneration

-

-

-

-

-

2/1.0

8/1.5

7/1.7

Mixed cell infiltrates

-

-

-

-

-

1/1.0

4/1.8

3/2.0

Transitional cell hyperplasia

-

-

-

-

-

2/2.0

1/1.5

4/1.8

C = control, LD = low dose, MD = medium dose, 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, ** = application of males and females with 240 mg/kg bw/day from premating day 9 onwards  

Histological changes were described, wherever possible, according to distribution, severity and morphologic character.

Severity scores were assigned from a scale of 1-5.

Grade 1, Minimal

This corresponds to a histopathologic change ranging from inconspicuous to barely noticeable but so minor, small, or infrequent as to warrant no more than the least assignable grade. For multifocal or diffusely-distributed lesions, this grade was used for processes where less than approximately10% of the tissue in an average high-power field was involved.

Grade 2, Slight

This corresponds to a histopathologic change that is a noticeable but not a prominent feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was used for processes where between approximately 10% and 25% of the tissue in an average high-power field was involved. 

Grade 3, Moderate

This corresponds to a histopathologic change that is a prominent but not a dominant feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was used for processes where between approximately 25% and 50% of the tissue in an average high-power field was involved. 

Grade 4, Marked

This corresponds to a histopathologic change that is a dominant but not an overwhelming feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was used for processes where between approximately 50% and 95% of the tissue in an average high-power field was involved. 

Grade 5, Severe

This corresponds to a histopathologic change that is an overwhelming feature of the tissue. For multifocal or diffusely-distributed lesions, this grade was used for processes where greater than approximately 95% of the tissue in an average high-power field was involved. 

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:

Note: The study record will be updated when the final report is available from the laboratory.

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 63 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 will be dosed after the mating period until the minimum total dosing period of 28 days is 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 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. 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. However, a slight tendency towards a lower mean total number of pups in the HD group on PND 0 may be test item-related. Mean number of CL and IS showed statistically significantly lower in the HD group and LD and MD groups showed a tendency towards lower CL and IS numbers.

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. 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. 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. Based on the observed effects of the test item on health condition, body weight development, food consumption, and kidney effects, effects on anogenital distance and estrous cyclicity could be based on general toxicity in the HD group.

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.

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

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. The study record will be updated when the final report is available from the laboratory.

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 63 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 will be dosed after the mating period until the minimum total dosing period of 28 days is 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 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. 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. However, a slight tendency towards a lower mean total number of pups in the HD group on PND 0 may be test item-related. Mean number of CL and IS showed statistically significantly lower in the HD group and LD and MD groups showed a tendency towards lower CL and IS numbers.

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. 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. 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. Based on the observed effects of the test item on health condition, body weight development, food consumption, and kidney effects, effects on anogenital distance and estrous cyclicity could be based on general toxicity in the HD group.

Based on the findings of this study the 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.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the 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 or for effects on or via lactation when the criteria outlined in Annex I of 1227/2008/EC are applied.