Registration Dossier

Administrative data

Description of key information

Subacute NOAEL (male/female, rat): 40 mg/kg bw/day (OECD 422/GLP)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Dose Range Finding Study for Reproduction / Developmental Toxicity Screening Test after Oral Administration in Wistar Rats (OECD 421/422)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
31 July 2018 - 07 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: DRF study in rats for OECD 421/422
Version / remarks:
The details in the study report results relevant to study number 183812 (Combined Repeated Dose Oral Toxicity Study with the Reproduction / Developmental Toxicity Screening Test in Wistar Rats with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)- 1,3,5-triazin-2-amine).
GLP compliance:
no
Remarks:
DRF study; GLP not required
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: 384 - 406 g (mean: 393.42 g, ± 20 % = 314.73 – 472.10 g); females: 228 - 256 g (mean: 238.92 g, ± 20 % = 191.13 – 286.70 g)
- Housing: Full barrier in an air-conditioned room; The animals were kept in groups of 3 animals / sex / cage in IVC polysulphoge cages (double decker, type GR1800) on Altromin saw fibre bedding (except during the mating period when one female was paired with one male and during gestation/lactation period when females were housed individually in type III H IVC polysulphone cages)
- 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 oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle was selected based on the test item’s characteristics. The test item formulations were prepared freshly on each administration day before the administration procedure. The test item were emulsified in corn oil. The test item were 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 it to a water 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 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): MKCD 1021
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
A dose formulation analysis was not performed in this study.
Duration of treatment / exposure:
The animals were treated with the test item formulation or vehicle on 7 days per week 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.
Frequency of treatment:
7 days per week
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
LD/low dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
MD/medium dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
HD/high dose, 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.
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
HD(high dose); 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.
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
HID/high intermediate dose. Due to the high mortality rate in the HD group and also after dose reduction from 1000 mg/kg body weight/day to 600 mg/kg body weight/day, an additional group was inserted and treated with 500 mg/kg body weight/day (HID) to investigate whether 500 mg/kg body weight/day is a feasible dose for higher tier studies.
No. of animals per sex per dose:
3 males
3 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In consultation with the sponsor the following doses were selected for the 3 dose groups (LD = low dose 100 mg/kg bw, MD = medium dose 300 mg/kg bw, HD = high dose 1000mg/kg bw) and 1 control group (C). Due to the high mortality rate in the HD group and also after dose reduction from 1000 mg/kg body weight/day to 600 mg/kg body weight/day, an additional group was inserted and treated with 500 mg/kg body weight/day (HID) to investigate whether 500 mg/kg body weight/day is a feasible dose for higher tier studies.
- Rationale for animal assignment (if not random): randomisation was performed with IDBS Workbook 10.1.2 software

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes; 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. Females showing signs of abortion or premature delivery prior to the scheduled scarification of the animals were sacrificed and subjected to a thorough macroscopic examination.
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.
- Time schedule: at least once a day

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups. Animals prematurely sacrificed were 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 weekly 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.


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table below)
The males were sacrificed after completion of the mating period on day 29 with the exception of animal nos. 10, 11, and 12 of the high dose group which were sacrificed during the premating phase due to animal welfare reasons (no.10 on PMD 7, no. 11 on PMD 3 and no. 12 on PMD 8). The females were sacrificed along with their pups on post-natal day 4 with the exception of animal nos. 22, 23, and 24 of the high dose group which were sacrificed due to animal welfare reasons during the premating phase (no. 22 and no. 23 on PMD 8 and no. 24 on PMD 7). Non-pregnant females were sacrificed on day 26 (female no. 17 of the LD group).

Dead pups and pups sacrificed on day 4 post-partum or shortly thereafter were carefully examined externally for gross abnormalities.

Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear as an evidence of mating.

All animals were subjected to a detailed gross necropsy which includes 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), 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 and then transferred in 70 % ethanol.

All macroscopic findings were recorded and organs showing gross abnormalities and all organs listed were preserved in 4 % neutral-buffered formaldehyde except testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70 % ethanol.

All animals found dead and/or intercurrently sacrificed were subjected to a gross necropsy.

The number of implantation sites and corpora lutea was recorded for each parental female at necropsy. If appropriate and possible, the number of corpora lutea and implantation sites was recorded for any females sacrificed 26 days after the end of the mating period with no evidence of mating and or on day 26 post-coitum due to non-delivery.

Organ Weights
Reproductive organs from all animals were weighed (testes, epididymides, prostateseminal vesicle with coagulating glands as whole, ovaries, uterus with cervix).
Organ weights from animals found dead or euthanized due to animal welfare were not taken.
Other examinations:
Litter Observations

The duration of the gestation was recorded and was calculated from day 0 of the pregnancy. Each litter was examined as soon as possible after 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 parturition (day 0 post-partum) and on day 4 post-partum. Live pups were identified by writing numbers on the back with the help of a permanent marker or by tattooing. In addition to the observations of parent animals, any abnormal behaviour of the offspring was recorded.
Statistics:
Mean and standard deviations
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 6, Table 7, Table 8, Table 9, Table 29 and Table 30.

Male no. 10 which was euthanized in a moribund condition on premating day showed the clinical signs of a stilted gait, diarrhea, sunken flanks, dehydration, piloerection, moderately reduced spontaneous activity, and nasal discharge between PMD 3 and PMD 7 (day of sacrifice).

Prematurely sacrificed HD male no. 11 was observed with signs of piloerection, diarrhea, sunken flanks, dehydration and a stilted gait on the day of sacrifice (PMD 3).

Male no. 12 of the HD group which was sacrificed due to animal welfare reasons on PMD 8 was observed with clinical signs of piloerection, diarrhea, sunken flanks, dehydration, stilted gait, chromodacryorrhea, and a bloody wound on the tail root between PMD 3 and PMD 8 (day of sacrifice).
Prematurely sacrificed HD female no. 22 showed the clinical signs of diarrhea, dehydration, sunken flanks, stilted gait, reduced spontaneous activity, hypothermia, and a bloody wound on the tail root between PMD 3 and the day of sacrifice (PMD 8).

Female no. 23 of the HD group was euthanized in a moribund condition with clinical signs of diarrhea, sunken flanks, stilted gait, dehydration, and hypothermia between PMD 3 and PMD 8 (day of sacrifice).

Prematurely sacrificed HD female no. 24 was observed with clinical signs of stilted gait, sunken flanks, diarrhea, dehydration, reduced spontaneous activity between PMD 3 and the day of sacrifice (PMD 7).

3/3 females of the MD group were observed with the clinical sign of sunken flanks on PMD 3. As this finding occurred on a single day it is not considered toxicologically relevant.

The clinical sign of moving the bedding was observed transiently in 2/3 males of the LD group, 3/3 males of the MD group, 2/3 males of the HD group and 1/3 females of the control group, 1/3 females of the LD group, 3/3 females of the MD group, and 3/3 females of the HD group. The clinical sign of increased salivation was seen in 1/3 males of the LD group, 3/3 males of the MD group, 2/3 males of the HD group, and1/3 females of the LD group, 3/3 females of the MD group, and 3/3 females of the HD group. As both signs were observed in short timely relation to test item administration or in anticipation thereof they were considered to be a sign of discomfort or a local reaction of the test item.

Low incidences of clinical signs like hairless areas, crusts, or wounds in few animals of the control or test item-treated groups were seen without dose dependency and were considered as incidental.

None of the females showed clinical signs of abortion or premature delivery.

The clinical signs in males of the HID group which were sacrificed prematurely included diarrhea, piloerection, reduced spontaneous activity, hypothermia, stilted gait, sunken flanks, hunched posture, dehydration, nasal discharge, and abnormal breathing.
Females were observed with clinical signs of piloerection, diarrhea, hunched posture, and reduced spontaneous activity in the second half of the treatment period.

The clinical sign of moving the bedding was observed transiently in 3/3 males and 3/3 females of the HID group. The clinical sign of increased salivation was seen in 2/3 females of the HID group. Both signs were observed in relation to test item administration or in anticipation thereof they were considered to be a sign of discomfort or a local reaction of the test item.
Mortality:
mortality observed, treatment-related
Description (incidence):
For a summary see Table 4 and Table 5.

During the treatment period of this study mortality occurred in 3/3 males of the HD group, 3/3 females of the HD group, and 3/3 males of the HID group.

Male no. 11 of the HD group was euthanized in a moribund condition on premating day 3. HD male no. 10 was sacrificed due to animal welfare reasons on premating day 7, and male no. 12 of the HD group was euthanized due to animal welfare reasons on premating day 8.

HD female no. 24 was sacrificed in a moribund condition on premating day 7 and female nos. 22 and 23 of the HD group were euthanized due to animal welfare reasons on premating day 8.

Male no. 25 was euthanized in a moribund condition on treatment day 9, male no. 26 was euthanized due to animal welfare reasons on treatment day 10, and male no. 27 was euthanized in a moribund condition on treatment day 28.

All remaining animals (control, LD, and MD) survived their 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 10 to Table 16 and Table 31 to Table 38.

Treatment with the test item had no toxicologically relevant effect on body weight development of male and female animals of the LD group during the whole treatment period of this study.

Mean body weight of males of the HD group was lower compared to the controls on premating day 7 (deviation of the HD group from the control group: 17%) and compared to the body weight on premating day 1. HD animals showed mean body weight loss of 57 g during the first week of treatment.

Mean body weights of the MD males were marginally lower when compared to the control group from premating day 7 until the end of the treatment period (mean body weight of the MD group was 3 % - 6 % lower compared to the mean body weight of the controls). Accordingly, males of the MD group showed a lower mean body weight gain compared to the controls throughout the treatment period (body weight gain of MD males was 34 % lower compared to the control group considering premating day 1 to postmating day 14).

Females of the HD group showed a lower mean body weight on premating day 7 when compared to the female control group (deviation from control: 13%) and premating day 1. HD females showed a mean body weight loss of 35% during the first week of treatment.

Mean values of body weigh were slightly lower in the females of the MD group when compared to the controls from premating day 14 onwards (mean body weight of MD females was 4 % - 12 % below mean body weight of the control group. Mean body weight gain was comparable between MD females and females of the control group with the exception of the second week of treatment (premating day 7 - premating day 14) where females of the MD group showed a mean body weight loss of 15 g compared to a mean body weight gain of 5 g in the control group.

Males of the HID group were observed with a lower mean body weight on day 7 compared to day 1 of body weight measurement (mean bw day 1: 427 g, mean bw day 7: 386 g). Females of the HID group showed a body weight loss during the first week of treatment (mean bw day 1: 223 g, mean bw day 7: 218).
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 18, Table 19, Table 39 and Table 40.

Mean food consumption of was not affected in males of the LD and MD group when compared to the control males throughout the premating period. Food consumption of HD males was lower compared to the control group during the first week of treatment (deviation of the HD group from controls: 84%). However, this value has to be considered carefully as food consumption was available from only 2 males from treatment day 3 onwards.

Mean food consumption of females was not affected by treatment with the test in the LD and MD group compared to the female control group throughout the treatment period (premating, gestation, and lactation). Females of the HD group showed lower mean food consumption compared to the controls during the first week of treatment (deviation of the HD group from the control group: 36%).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 27 and Table 48.

There were no test item-caused differences in the weight of reproductive organs between treated groups and the respective control group.
However, mean prostate weight of LD males was slightly lower compared to controls (deviation from control: 20%). Due to the lack of dose dependency this trend was not considered test item-related.

Mean ovary weight was shown to be slightly higher in the females of the LD (deviation from control: 18%) and MD (deviation from control: 21%) group compared to the mean ovary weight of the control females. Mean uterus weight was slightly higher in LD females compared to the control group (deviation from control: 13%). However, these effects are not considered toxicologically relevant as female reproductive organs may undergo changes depending on the stage of the estrous cycle.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 26.

Macroscopic findings that could be attributed to treatment with the test item consisted of an abnormal colored stomach (white and spotted) of male no. 11 which was euthanized due to animal welfare reasons and a stomach containing few (2-6) masses of male no. 12 which was euthanized in a moribund condition.

Single findings of an enlarged spleen in 1/3 females of the control group, a thymus with several (7-12) red foci in 1/3 males of the MD group, a small thymus in 1/3 females of the MD group, kidneys with an abnormal surface (crater) in 1/3 females of the MD group were within the range of normal background alterations and thus are not considered toxicologically relevant.

Male no. 26 of the HID group which was euthanized in a moribund condition on treatment day 10 was observed with a fluid filled (clear) and dark/white spotted stomach.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Litter Data
For a detailed description of the findings see Table 20 and Table 41.
Treatment with the test item had no toxicologically relevant effects on pup associated parameters including total number of pups, still births, runts, and sex ratio.
Litters were delivered by all dams of the control and the MD group and by 2/3 females of the LD group. Treatment with the test item had no effect on the total number of pups when comparing treated groups with the control group on PND 0 and PND 4.
There were no considerable differences in the sex ratio between test item-treated groups and the control group. No runts and still births were observed in any of the test item-treated groups or the control group.

Litter Weight Data
For a detailed description of the findings see Table 21 and Table 42.
Treatment with the test item had no toxicologically relevant effect on litter weight data.
Mean pup weight was comparable between pups of the test item-treated groups (LD and MD) and the controls.
In accordance with a slightly lower number of live male pups in the MD group on PND 0 and PND 4 male litter weight and total litter weight was slightly lower in the MD group when compared to the controls. However, this was not considered toxicologically relevant.

Precoital Interval and Duration of Gestation
For a detailed description of the findings see Table 22 and Table 43.
Treatment with the test item had no toxicologically relevant effect on precoital interval and the duration of gestation.
Mean precoital interval was slightly lower in the MD group when compared to the controls. However, this variation is within the normal range of variation. Mean duration of gestation was comparable between test item-treated groups and the control group.

Pre- and Post-Natal Data
For a detailed description of the findings see Table 23 and Table 44.
Treatment with the test item had no toxicologically relevant effect of pre- and postnatal data in this study. Mean values of corpora lutea, implantation sites, alive pups on PND 0 and PND 4, pre-implantation loss, and post-implantation loss. Differences were within the normal range of variation of this strain.

Reproductive Indices
For a detailed description of the findings see Table 24 and Table 45.
There were no treatment-related effects on the reproductive indices including copulation index, fertility index, delivery index and viability index when comparing the LD and the MD group with the control group.
Slightly lower fertility index of the LD group (66.67 %) compared to the control group was based on one non-pregnant female of the LD group. This was considered incidental and thus not toxicologically relevant.
Apart from the fertility index of 66.67 % in the LD group all indices were 100 % in the control, the LD, and MD group.

Pup Survival Data
For a detailed description of the findings see Table 25 and Table 46.
Survival of live born pups was not different between the control group and groups treated with the test item. All live born pups survived from PND 1 to PND 4 in all groups.

Pup External Findings
For a detailed description of the findings see Table 47.
One pup of the control group (dam no. 14, pup no. 3) was observed to be pale on PND 1. As this finding occurred in the control group it is not considered toxicologically relevant. There were no further external macroscopic findings in any of the pups of the control or the test item-treated groups.
Dose descriptor:
other: DRF study
Effect level:
0 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: DRF study
Critical effects observed:
no
Conclusions:
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.
Executive summary:

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; 183812) with 4,6-dichloro-N-(1,1,3,3-tetramethylbutyl)-1,3,5-triazin-2-amine.

Endpoint:
repeated dose toxicity: oral, 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 - 2019
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 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:
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
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. 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 mid 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).
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.

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, 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 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.
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, treatment-related
Description (incidence and severity):
For a detailed description of the findings see Table 29, Table 38, Table 58 and Table 71.

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.

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, 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:
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
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
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

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 (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:

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40 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.
System:
urinary
Organ:
kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

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.

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.

Additional information

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) is classified for specific target organ toxicity category 2 (repeated; oral (kidney)) when the criteria outlined in Annex I of 1272/2008/EC are applied.