N-ethylpropylamine

Endpoint summary

Description of key information

oral: NOAEL (male/female) = 600 mg/kg bw/day (rat; BASF SE 2022)
dermal: no data availble
inhalative: no data availble

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb. - Oct. 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): BASF SE
- Lot/batch number of test material: B987 – 20210115
- Purity: content: 87.4 +/- 0.3 g/100 g (water: 10.8 g/100 g)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at controlled room temperature (15-25℃, ≤70% relative humidity)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: The test item was considered stable when stored under appropriate storage conditions. All test item formulations were shown to be homogeneous in dose formulation analyses. Formulations were considered to be adequately stable under the study conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): The test item was neutralized with hydrochloric acid (target pH 6.5-7.5) after being formulated in the selected vehicle distilled water, as a visibly stable homogenous emulsion at the appropriate concentrations according to the dose level and volume selected in the test facility. Formulations were prepared daily.
- Final concentration of a dissolved solid, stock liquid or gel: 10, 30, 100/60 mg/mL


FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in distilled water

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, (Address: Sandhofer Weg 7, D-97633, Sulzfeld, Germany) from SPF colony.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: males: 361-411 g, females: 223-270 g (at the start of the treatment)
- Fasting period before study: no
- Housing: Rodents were group-housed, 2 animals of the same sex and dose group/cage, with the exception of the mating and gestation,
delivery, lactation period, when they were paired or individually housed (with pups), respectively. Males were individually housed after their mating finished until the end of the mating period.
- Diet (ad libitum): ssniff® SM R/M “Autoclavable complete diet for rats and mice – breeding and maintenance”
- Water (ad libitum): tap water from the municipal supply
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.9-24.9
- Humidity (%): 25-66
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 March 2022 To: 15 May 2022

Route of administration:

oral: gavage

Details on route of administration:

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, in each morning, by oral gavage using a tipped gavage needle attached to a syringe (the surface of the needle was carefully cleaned before every dosing). A constant volume of 10 mL/kg bw was administered to all animals. The actual volume to be administered was calculated and adjusted based on each animal’s most recent body weight.

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 10, 30, 100/60 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 2201-8186 / 2203-8230

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Sample collection was performed at a total of three occasions (during the first and last weeks and approximately midway during the treatment period).
- On each sampling occasion, top, middle and bottom duplicate samples were taken from test item formulations for concentration and homogeneity measurement. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
- Analysis of the formulations for concentration and homogeneity of test item was performed using a validated analytical HPLC-UV (High Performance Liquid Chromatography with UV detector) method
- The measured concentrations of the test item in the different formulations varied between 91.9% and 99.2% of the nominal concentrations. The results were considered to be acceptable.

Duration of treatment / exposure:

- males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period)
- females were dosed for 14 days pre-mating, for up to 14 days mating period, then the animals were treated through gestation and up to and including the day before necropsy (13 days postpartum dosing)

Frequency of treatment:

daily on a 7 days/week basis

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

reduced to 600 mg/kg bw/d on Day 7

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on
the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the high dose for this study, this level was reduced to 600 mg/kg bw/day on Day 7 based on the observed effects.
- Fasting period before blood sampling for clinical biochemistry: yes, overnight
- Dose range finding studies: Four male and four female animals per group were treated daily for 14 consecutive days in order to obtain preliminary information on the potential toxicity of the test item following repeated administration at 3 dose levels (0, 300, 1000 mg/kg bw/day). The control group was treated with the vehicle only. Parameters analysed / observed were mortality/morbidity checks, clinical observations, cage side observations, detailed clinical observations, body weights, food consumption, necropsy, organ weights, statistical analysis. None of the changes observed were of a severity to preclude 1000 mg/kg/day being used in the subsequent OECD 422 study.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: check for signs of morbidity and mortality twice daily (at the beginning and end of each working day); general clinical observations were made once a day in the afternoon (when signs of toxicity were observed in the mid and high dose groups, the animals were observed more frequently, three times daily (from Day 3 to Day 10)
- Cage side observations: signs of morbidity and mortality; general (routine) clinical observations (not further specified)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure on Day 0 (to allow for within-subject comparisons), then weekly and on the day of necropsy in the morning or before treatment at approximately the same time (with minor variations as practical during the working day)
- Detailed clinical observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lacrimation, piloerection, pupil size and unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self- mutilation, walking backwards), observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma; pertinent behavioural changes, all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On Gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
Furthermore, mated females were examined carefully around the time of expected delivery for any signs of difficult or prolonged parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0, and afterwards weekly, and at termination
Parental females were weighed at least on Gestation Day (GD) 0, 3, 7, 10, 14, 17 and 20, on PPD (Post-partum Day) 0 (within 24 hours after parturition), 4, 7, 10 and 13, and at termination. The body weight of the female animals measured on GD3, GD10 and GD17 as well as PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data are not evaluated statistically.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: on body weight measurements day; Food consumption was measured more frequently during the gestation period (at least on GD 0, 3, 7, 10, 14, 17 and 20) and lactation period (at least on PPD 0, 4, 7, 10 and 13).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter had been terminated)
- How many animals: 7 males and 5 females/group (randomly selected)
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled necropsy
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter had been terminated)
- How many animals: 7 males and 5 females/group (randomly selected)
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes (thyroid hormone analysis)
- Time of blood sample collection: on the day of termination
- Animals fasted: Yes (overnight period of food deprivation, in case of females this happened after the litter had been terminated)
- How many animals: all dams and non-pregnant adult females, all adult males

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (for example, males on Day 23 am, females on PPD 7 am); 5 animals/sex/dose (not the same animals as selected for clinical pathology)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera)

HISTOPATHOLOGY: Yes (see table 3)
- detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as in table 3) in the control and high dose groups: selected 5 main animals/sex/group (the same animals were used for clinical pathology or neurology examination),
• all macroscopic findings (abnormalities), except of minor order from all animals,
• on the retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation
gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the control and
high dose groups, and of all males that failed to sire and all females that failed to deliver healthy pups.
- Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
- Special attention was paid to the organ weight, appearance and histopathology of immune system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes, bone marrow).
- Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.

Statistics:

see "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- abnormal skin colour in 9/12 males and 9/12 females from Day 6 to Day 14. The longevity of the symptoms was 7 days.
- slightly to extremely decreased activity in all animals from Day 0 to Day 9. The longevity of the symptom was 7 days.
- slight to moderate ataxia in all animals from Day 3 to Day 10. The longevity of the symptom was 8 days.
- partially closed eyes (both eyelids) in all males and 11/12 females from Day 2 to Day 9. The longevity of the symptom was 6 days.
- hunched back in all animals from Day 1 to Day 14. The longevity of the symptom was 8 days.
- piloerection in all animals from Day 1 to Day 10. The longevity of the symptom was 8 days.
- incoordination (slight to extreme) in 11/12 males and 11/12 females from Day 0 to Day 2. The longevity of the symptom was 3 days.
- prostration in 5/12 males and 4/12 females from Day 1 to Day 8. The longevity of the symptom was 2 days.

Due to these signs the high dose was reduced from 1000 mg/kg bw/day to 600 mg/kg bw/day
after 7 Days treatment.

- red discharge (nose) in 1/12 males and 6/12 females from Day 21 to Day 49. The longevity of the symptom was 5 days.
- intermittent tremor in 6/12 males and 7/12 females from Day 2 to Day 8. The longevity of the symptom was 1 day.
- tonic convulsion was observed in 1/12 males and 2/12 females from Day 1 to Day 5. The longevity of the symptom was 3 days.
- lethargy in 2/12 males on Day 6.

The above treatment related clinical signs were seen early in the study, exclusively in the high
dose, they recovered soon after the high dose level was reduced from 1000 to 600 mg/kg/day.
The abnormal skin colour was generally on the paws, ears, tails and nose, which did not wash
off (it was within the skin). The test item was not coloured.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- weight loss during the first week: High dose males lost weight in the first 7 days, to be 20% below controls. Thereafter, statistically significantly lower body weight continued from Day 7 to Day 27 (from -20.0% to -6.9%, p<0.01). High dose females lost weight and were ~12% below control on Day 7 but were similar to Control by Day 14
- statistically significantly increased body weight gain in males from Day 7 to Day 21 (compensation for the initial weight loss); the overall body weight gain was decreased by -33.1%, (p<0.01)
- statistically significantly increased body weight gain in females from Day 7 to Day 14 (compensation for the initial weight loss; by 249.5%, p<0.01) with high dose and controls having similar weights by Day 14
- statistically significantly decreased body weight gain in females in the period GD 14 to GD 20 (by -18.7%, p<0.05), but the overall body weight gain was considered to be normal

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- significantly lower food consumption of males (by approximately -17.8%, p<0.01) when compared to the control during the whole study
- reduced food consumption of females in the first two weeks (by approximately -26.3%, p<0.05) compared to control

The changes were reflected by body weight, as a treatment effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly higher red cell distribution width (by 12.3%, p<0.01) in males; The data were slightly outside of the historical control range but without any consequence in other RBC parameters, the difference was considered not to be adverse.
- statistically significantly lower PTT (by -3.9%, p<0.05) in males; The data were within the historical control range and all data was considered to be normal, hence it was not considered as test item related effect.
- statistically significantly lower eosinophil relative counts (by -74.3%, p<0.05) in females; The data were slightly below the historical control range, but all animals were considered to be normal and there was no histopathological relevance; it was not considered as an adverse test item related effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly increased potassium level (by 15.3%, p<0.05) and decreased albumin
level (by -7.7%, p<0.01) in males; The data were within the historical control range and without histopathological relevance, it was not considered as test item related effect.
- statistically significantly increased cholesterol level females (by 31.4%, p<0.01) and males (by 49.0%, p<0.01); The data were outside of the historical control range for the males but without histopathological relevance, it was considered as test item related but not adverse effect.
- statistically significantly decreased A/G ratio (by -13.0%, p<0.01) in females; The data were within the historical control range and without histopathological relevance, it was not considered as test item related effect.

Mid dose group:
- statistically significantly increased ALT/GPT (by 55.4%, p<0.01) in females, with no dose response; All data were in the normal historic control range. It was not considered as test item related effect.

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly decreased terminal body weight (by -7.2%, p<0.01) in males compared to control
- the adrenal glands appeared to be enlarged in males with statistically significantly increased body related weight (by 21.7%, p<0.05); The data were slightly above the historical control range but without any histopathological relevance, it was not considered as an adverse test item related effect.
- statistically significantly increased absolute, body and brain related weights of the kidney (by
10.5, 19.0 and 13.6%, respectively, p<0.01) in males; All the data were within the historical control range and there were no histological changes. This was considered as not adverse.
- statistically significantly increased absolute, body and brain related weights of the kidney (by ~16% (p>0.01), relative to body weight) in females; All the data were above the historical control range but without histopathological relevance, it was not considered as an adverse test item related effect.
- statistically significantly decreased absolute, body and brain related weights of the seminal
vesicles (by 20.4, 14.5 and 18.1%, respectively) in males; The High dose were within the historical control range when adjusted for body weight. These changes did not have histopathological relevance but low weights are common in males with low food intake in studies. The change was not considered as adverse.
- statistically significantly increased absolute, body and brain related weights of the adrenal
gland (by 25.9, 25.4 and 30.3%, p<0.01) in females; All the data were outside of the historical control range but without histopathological relevance, it was not considered as an adverse test item related effect.
- statistically significantly increased absolute, body and brain related weights of the liver (by ~13% (p<0.01), relative to body weight) in females. The data were outside of the historical control range but without histopathological relevance. Enlarged livers are common with exposure to xenobiotics, but hypertrophy of less than ~15% liver weight change is commonly not visible at
histopathology when it is an adaptive change. It was not considered as an adverse test item
related effect.

Mid dose group:
- statistically higher adrenal weight in males when adjusted for brain weight, but without a clear dose response
- significantly higher (p>0.05) male kidney weights adjusted for brain weight; All the data were within the historical control range and there were no histological changes. This was considered as not adverse.
- statistically significantly increased absolute, body and brain related weights of the kidney (by ~10% (p>0.05), relative to body weight) in females; All the data were above the historical control range but without histopathological relevance, it was not considered as an adverse test item related effect.
- statistically significantly increased absolute, body and brain related weights of the liver (by ~9% (p<0.01), relative to body weight) in females. Enlarged livers are common with exposure to xenobiotics, but hypertrophy of less than ~15% liver weight change is commonly not visible at
histopathology when it is an adaptive change. It was not considered as an adverse test item
related effect.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: based on recovery for adverse effects at 1000 mg/kg/day in clinical observations, body weight and food consumption results of the high dose group

Key result

Critical effects observed:

no

Conclusions:

Based on the existing results of this study, the following No-Observed-Adverse-Effect Level (NOAEL) was considered:

The NOAEL for systemic toxicity of the parental generation: 600 mg/kg bw/day (based on recovery for adverse effects at 1000 mg/kg/day in clinical observations, body weight and food consumption results of the high dose group).

Executive summary:

The purpose of this study was to obtain information on the possible toxic effects of the test substance following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (distilled water). The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum.
The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the high dose for this study, this level was reduced to 600 mg/kg bw/day on Day 7 based on the observed effects.

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation and clinical chemistry). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The T4 and TSH levels in the PND (Post-natal Day) 13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the control and high dose groups, and reproductive organs of all high dose and control animals and all those male / female mating pairs where no liveborn pups were achieved. Dosing formulations were analysed for concentration and/or homogeneity on three occasions during the study. Overall, the formulations were considered adequate for the study.

A daily oral gavage administration of the test substance to Wistar rats for 28 days in males and 50-54 days in females did not cause mortality, but significant adverse effects >MTD were seen in the high dose in the first week, hence the dose for this group was reduced from 1000 to 600 mg/kg/day on Day 7 (animals were generally recovered by about Day 14). Test item related clinical signs (closed eyes, red discharge, intermittent tremor, lethargy, ataxia, incoordination, tonic convulsion, decreased activity, prostration, abnormal skin colour of extremities, hunched back and piloerection) were observed during the first two weeks in the high dose groups. Thereafter, and in low and mid groups, there were no adverse treatment related clinical signs.
Body weight losses of ~20% in the high dose animals in the first week, with low food intake, was a transient test item related effect; by Day 14 after reduction of the dose level, males were only ~11% below control and females regained and had the same as weight controls. Although 1000 mg/kg/day was >MTD, thereafter at 600 mg/kg/day there were no adverse body weight effects.
At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli ascribed to a test item effect.
No test item-related adverse effects were seen in the clinical pathology parameters; cholesterol levels appeared to be elevated in the high dose of both sexes, but in the absence of any other changes in the animals including histopathology, this was not considered as a clear adverse effect.
No adverse test item-related organ weight changes were observed; increases in liver and kidney weights, without any histology changes, were considered as most probably adaptive, higher adrenal weights without histology changes, were considered not adverse. No macroscopic or microscopic treatment related findings were recorded during the histopathology evaluation.
No microscopic changes were observed in the reproductive organs of the non-pregnant females and their male mating pairs.
No statistically significant or biologically relevant changes were observed in the T4 or TSH hormone concentration in the test item treated parental males. No relevant changes were noted in the absolute thyroid or relative (to body) thyroid weights of male parental animals in any dose groups.
No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Available data is reliable and sufficient for assessment.

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

Additional information

Oral:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD TG 422 and GLP, the test substance was administered by oral gavage to male and female Wistar rats at 3 dose levels (100, 300, 1000/600 mg/kg bw/d). A control group received the vehicle only (distilled water). The dose levels were selected by the Sponsor in consultation with the Study Director based on the results of a Dose Range Finding (DRF) study. Based on those results, 1000 mg/kg bw/day was selected as the high dose for this study, this level was reduced to 600 mg/kg bw/day on Day 7 based on the observed effects.

Parameters measured during the study included twice a day mortality checking, daily routine and weekly detailed observation of clinical signs, weekly body weight and food consumption measurements and clinical pathology evaluation (including haematology, coagulation and clinical chemistry). Neurological assessment (Functional Observation Battery (FOB) including measurements of the landing foot splay, grip strength as well as locomotor activity measurement) was performed during the last week of the treatment for each sex. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination (Day 28 for males, PPD (Post-partum Day) 14 for females), necropsy with macroscopic examination was performed. Weights of selected organs were recorded, and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals or F1 animals. The T4 and TSH levels in the PND (Post-natal Day) 13 pups and parental males were also determined. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 animals/sex in the control and high dose groups, and reproductive organs of all high dose and control animals and all those male / female mating pairs where no liveborn pups were achieved. Dosing formulations were analysed for concentration and/or homogeneity on three occasions during the study. Overall, the formulations were considered adequate for the study.

A daily oral gavage administration of the test substance to Wistar rats for 28 days in males and 50-54 days in females did not cause mortality, but significant adverse effects >MTD were seen in the high dose in the first week, hence the dose for this group was reduced from 1000 to 600 mg/kg/day on Day 7 (animals were generally recovered by about Day 14). Test item related clinical signs (closed eyes, red discharge, intermittent tremor, lethargy, ataxia, incoordination, tonic convulsion, decreased activity, prostration, abnormal skin colour of extremities, hunched back and piloerection) were observed during the first two weeks in the high dose groups. Thereafter, and in low and mid groups, there were no adverse treatment related clinical signs.
Body weight losses of ~20% in the high dose animals in the first week, with low food intake, was a transient test item related effect; by Day 14 after reduction of the dose level, males were only ~11% below control and females regained and had the same as weight controls. Although 1000 mg/kg/day was >MTD, thereafter at 600 mg/kg/day there were no adverse body weight effects.
At the functional observation battery (FOB) and locomotor activity measurement, there were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli ascribed to a test item effect.
No test item-related adverse effects were seen in the clinical pathology parameters; cholesterol levels appeared to be elevated in the high dose of both sexes, but in the absence of any other changes in the animals including histopathology, this was not considered as a clear adverse effect.
No adverse test item-related organ weight changes were observed; increases in liver and kidney weights, without any histology changes, were considered as most probably adaptive, higher adrenal weights without histology changes, were considered not adverse. No macroscopic or microscopic treatment related findings were recorded during the histopathology evaluation.
No microscopic changes were observed in the reproductive organs of the non-pregnant females and their male mating pairs.
No statistically significant or biologically relevant changes were observed in the T4 or TSH hormone concentration in the test item treated parental males. No relevant changes were noted in the absolute thyroid or relative (to body) thyroid weights of male parental animals in any dose groups.
No indication of an endocrine disruptor effect was observed in the study based on collected oestrus cycle data, anogenital distance measurement, nipple retention, thyroid hormone measurement, thyroid weight and histopathology and external reproductive organs analysis.

Based on recovery for adverse effects at 1000 mg/kg/day in clinical observations, body weight and food consumption results of the high dose group, the NOAEL for systemic toxicity was 600 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. The NOAEL determined in a rat study according to OECD TG 422 is 600 mg/kg bw/d (highest tested dose after reduction of the dose level from 1000 mg/kg bw/d which turned out to exceed the MTD). As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) 2020/1182.