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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

276-309-1

EC Name:

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

Cas Number:

72058-41-4

Molecular formula:

C11H18Cl2N4

IUPAC Name:

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

Test material form:

solid

Details on test material:

SMILES:
CC(C)(C)CC(NC1=NC(Cl)=NC(Cl)=N1)(C)C

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.

Test animals

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral 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

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

LD (low dose)

Doses / concentrations 2

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

MD (mid-dose)

Doses / concentrations 3

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

Doses / concentrations 4

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

Examinations

Observations and examinations performed and frequency:

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. 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.
OPHTHALMOSCOPIC EXAMINATION: Yes; see functional observations

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).

OTHER:
Functional Observations
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests.
Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes; 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.

The wet weight of the organs (Table 7) of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of the animal euthanised for animal welfare reasons were not recorded. Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.

- HISTOPATHOLOGY: Yes; 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. Thyroid/parathyroid glands from the remaining non-selected adult animals were not examined as thyroid/parathyroid glands from selected males and females showed no toxicologically relevant microscopic findings.

A full histopathology was carried out on the preserved organs and tissues of the animal which was euthanised due to morbidity.

In the control group kidneys from five selected males and females were examined. In the HD group, kidneys were examined from 10/10 males and 10/10 females (1/10 decedent female and 9/10 surviving females. Examination of the kidneys was extended to selected animals of the low dose and medium dose groups (five males and females from the low dose and the medium dose group, each) as treatment-related changes were observed in the kidneys of the high dose group.

Any gross lesion macroscopically identified was examined microscopically in all animals. Discoloration possibly due to the test item was evaluated in the organs of all dose groups.

Statistics:

A statistical assessment of the results of body weight and food consumption was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry 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. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.3.4 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

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.

Mortality:

mortality observed, treatment-related

Description (incidence):

For a summary see Table 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.

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, non-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 and male or female PND 13 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, Table 59 and Table 60.

Endocrine findings:

no effects observed

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, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 29, Table 38, Table 58

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 %); however, 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.

Absolute mean thymus weight was statistically significant lower in females of the MD (deviation from control: 25 %, p<0.05) and HD (deviation from control: 30 %, p<0.01) group compared to control females. Differences in mean thymus weight may be stress-induced. Without correlation of histological findings this was not considered toxicologically relevant.

Relative mean spleen weight was statistically significantly higher in males of the HD group compared to the control males (deviation of the HD group from the control group: 18 %, p<0.05). Relative mean adrenal gland weight was slightly higher in males of the HD group compared to control males without achieving statistical significance (deviation from control: 19 %). 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 any organ.

Slightly higher relative mean thyroid/parathyroid weight of females of the MD group compared to control females (deviation from control: 18 %) was not assumed relevant as HD animals were not affected.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Table 37.

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

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:

effects observed, treatment-related

Description (incidence and severity):

For a detailed description of the findings see Appendix 3 and Table 9 below.

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.

All other microscopic findings recorded in the surviving animals were within the range of background lesions which may be recorded in animals of this strain and age and in this study type

Histopathological findings: neoplastic:

not examined

Effect levels

Dose descriptor:

NOAEL

Effect level:

40 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

120 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

Based on the findings of this combined repeated dose and reproduction/developmental toxicity screening test in Wistar rats, the NOAEL (male/female) of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine for general toxicity is considered to be 40 mg/kg bw/day, based on the test item-related nephropathy effects.

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 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 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.

Based on the findings of this study the NOAEL (male/female) of 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine for general toxicity is considered to be 40 mg/kg bw/day.