N-ethylpropylamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

oral: NOAEL (male, female)systemic & reproductive effects = 600 mg/kg bw/d (OECD 422; rat; oral; BASF SE 2022)

dermal: no data available

inhalative: no data available

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

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

Vehicle:

water

Details on 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

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occurred, for up to 5 days
- Proof of pregnancy: the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines)
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): single

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

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks (after the animals had attained full sexual
maturity)

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

Parental animals: Observations and examinations:

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).

WATER CONSUMPTION: No

OTHER:
Neurological assessment: Functional Observational Battery and locomotor activity measurement
- 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)
- Parameters: functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength; body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation

Oestrous cyclicity (parental animals):

Oestrus cycles were monitored for all females by vaginal smears daily during the pre-exposure period before the treatments start. Any females that fail to show a 4-5 days cycle were not included in the study. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating (during the pre-mating and mating periods).
Additionally, vaginal smears were prepared and examined for each surviving female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight; epididymis weight; stages of spermatogenesis in the male gonads; histopathology of interstitial testicular cell structure, epididymis (left and right), seminial vesicles, testis (left and right)

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups), presence of gross anomalies, physical or behavioural abnormalities, anogenital distance (AGD), weight gain, postnatal mortality, presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

Postmortem examinations (parental animals):

SACRIFICE
- male animals: all surviving animals, one day after the last treatment
- maternal animals: all surviving animals, one day after the last treatment

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.
- detailed histological examination was performed as follows:
• on the selected list of retained tissues and organs (as in table 1) 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.

CLINICAL PATHOLOGY
- All animals including the selected animals for blood sampling were fasted (overnight period of
food deprivation, in case of females this happened after the litter had been terminated).
- Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior
to scheduled necropsy.
- Before necropsy blood was taken for thyroid hormone analysis by venipuncture (using vena sublingualis in case of adult animals) or decapitation (in case of pups) (from up to two pups per litter on PND4 (if possible); from all dams on PPD 14 and at least two pups per litter on PND13 (if possible); from all non-pregnant adult females on the day of termination; from all adult males on the day of termination). The collected pup blood (serum) samples were pooled by litter.
- The parameters indicated in Table 2 and 3 were evaluated.

Postmortem examinations (offspring):

Dead pups and pups terminated on PND4 and/or PND13 were carefully examined externally for gross abnormalities. After the external observation, the sex determined at birth was confirmed by observation of the internal reproductive organs, if possible. Presence of nipples/areolae in the PND13 male pups was also recorded.

Organ weights: Thyroid glands from one male and one female PND13 pup from each litter were weighed

Statistics:

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

Reproductive indices:

- Male Mating Index: measure of male’s ability to mate
- Female Mating Index: measure of female’s ability to mate
- Male Fertility Index: measure of male’s ability to produce sperm that can fertilise eggs
- Female Fertility Index: measure of female’s ability to become pregnant
- Gestation Index: measure of pregnancy that provides at least one live pup

for formulas see "Any other information on materials and methods icl. tables"

Offspring viability indices:

- Survival Index (survival index on PND13 was calculated from number of pups after culling on PND4 instead of number of pups born)
- Pre-implantation mortality
- Intrauterine mortality
- Total mortality
- Sex ratio

for formulas see "Any other information on materials and methods icl. 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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- pseudopregnancy in 10/12 females; As all mated normally with no delay in becoming spermpositive, and mean cycle lengths were normal, this was not considered to be an adverse
effect of the test item (but may have been related to the >MTD effect of treatment in the first 7
days).

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Three non-pregnant females (one mid dose and 2 high dose animals) were observed in the study. Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs. No microscopic changes were seen in these females or in their male pairs.

Key result

Dose descriptor:

NOAEL

Remarks:

for systemic toxicity

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

Dose descriptor:

NOAEL

Remarks:

for reproductive effects

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Key result

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- cold to touch (whole body) observed for 6 pups
- abnormal skin colour (whole body, blue or white) observed for 10 pups

Mid dose group:
- cold to touch (whole body) observed for 5 pups
- abnormal skin colour (whole body, blue or white) observed for 5 pups

Low dose group:
- cold to touch (whole body) observed for 3 pups

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly increased postnatal mortality % on PND4 (p<0.05) (within the historical control range) and thus a lower survival index at Day 4

Mid dose group:
- statistically significantly increased postnatal mortality mean and postnatal mortality % on PND4 (p<0.05) and thus a lower survival index at Day 4

Since there was no dose response and the high dose group was in the normal range, it was not considered as test item related effect.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly lower body weight on PND13 (by -14.8%, p<0.05) and body weight gain between PND4-13 (by -15.6%, p<0.05); The data were within the historical control range therefore, it was not considered as test item related effect.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- male ADG not adjusted for body weight was slightly below the controls; It was within the historical
control range and not considered as test item related effect.

Low dose group:
- statistically significantly increased AGD per litter in female pups; As it was not part of a dose response and the data were within the historical control range, it was considered as unrelated to treatment.

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly lower T4 hormone levels on PND13 (by -14.4%, p<0.01) when compared to the relevant control level; The data were outside of the historical control range and with dose response it was considered as test item related effect but without histopathological findings and organ weight changes it was not considered as adverse.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: no adverse effects observed up to and including the highest tested dose

Key result

Critical effects observed:

no

Reproductive effects observed:

no

Table 4: Summary of reproductive parameters (males)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of males used for mating	12	12	12	12
Number of successful mating	12	12	12	12
Number of infertile animals	0	0	0	0
Male mating index (%)	100	100	100	100
Male fertility index (%)	100	100	100	100

Table 5: Summary of reproductive parameters (females)

Parameters	Dose groups (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of females used for mating	12	12	12	12
Number of sperm positive females	12	12	12	12
Number of females with no implantation sites	0	0	1	2
Number of pregnant females	12	12	11	10
Number of pregnant females with live born(s)	12	12	11	10
Number of pregnant females with no live born	0	0	0	0
Female mating index (%)	100	100	100	100
Female fertility index (%)	100	100	92	83
Female gestation index (%)	100	100	100	100

Table 6: Summary of the pregnancy evaluation

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated females	12	12	11	10
Number of pregnant females	12	12	11	10
Duration of pregnancy (days)	22.25	22.33	22.82*	22.20	D
Number of implantations, mean	16.67	16.58	15.33	12.82	NS
Number of pups born, mean	14.50	14.50	15.09	12.60	NS
Number of live born pups, mean	14.50	13.58	14.45	12.20	NS
Pre-natal mortality, mean	2.17	3.00	2.27	1.90	NS
Pre-natal mortality (%), mean	12.97	17.76	13.96	15.78	NS
Post-natal mortality, mean PND0-4	0.25	0.17	3.09*	1.30	U
Post-natal mortality (%), mean PND0-4	1.53	1.90	19.02*	13.30*	U
Total mortality, mean PND4	2.42	3.17	5.36	3.20	NS
Total mortality (%), mean PND4	14.48	18.84	31.71	26.23	NS

NS: Statistically not significant when compared to the control.
D: Duncan’s multiple range test, U: Mann-Whitney U - test versus Control,
Statistical significance compared to control: * = p<0.05

Table 7: Summary of survival (offspring)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Post-natal mortality on PND0-13, mean	0.33	0.25	3.36	1.40	NS
Post-natal mortality on PND0-13 (%), mean	2.23	2.30	20.63	14.07	NS
Total mortality on PND13, mean	2.50	3.25	5.64	3.30	NS
Total mortality on PND13 (%), mean	15.00	19.39	33.26	26.94	NS
Survival index on PND0	100.00	92.11	95.37	96.11	NS
Sex ratio (%) on PND0 Female	53.33	43.93	55.91	52.16	NS
Survival index on PND4	98.47	98.25	80.98*	86.70*	U
Sex ratio (%) on PND4 Female	53.14	44.89	60.76	51.57	NS
Survival index on PND13	99.17	99.17	90.00	99.00	NS
Sex ratio (%) on PND13 Female	50.46	47.96	57.68	49.78	NS

NS: Statistically not significant when compared to the control
Sex ratio means the percentage of female pups
U: Mann-Whitney U-test versus control; Statistical significance compared to control: * = p<0.05, ** = p<0.01

Table 8: Summary of the offspring mortality

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Number of pups born	174 / 12	174 / 12	166 / 11	126/ 10	NS
Number of cannibalized pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of autolyzed pups	0 / 0	11 / 3	7 / 4	4 / 3	NA
Number of stillborn pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of live born pups	174 / 12	163^** / 12	159^** / 11	122^** / 10	CH
Number of found dead pups (born alive)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of living pups on PND0	174 / 12	163 / 12	159 / 11	122 / 10	NA
Number of cannibalized pups (PND0-13)	2 / 2	2 / 2	33 / 6	4 / 3	NA
Number of autolyzed pups (PND0-13)	2 / 2	1 / 1	4 / 2	9 / 5	NA
Number of found dead, intact pups (PND0-13)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Total number of pups died (born alive)	4 / 4	3 / 3	37 / 6	13 / 6	NS
Culled for blood sampling on PND4	21 / 11	20 / 10	14 / 7	12 / 7	NA
Culled pups on PND4	30 / 9	32 / 10	13 / 4	14 / 4	NA
Number of viable pups on PND13	119 / 12	108 / 12	95 / 11	82 / 10	NS

CH: Chi Square test, NS: Statistically not significant when compared to the control,
NA: Not applicable. Statistical significance compared to control: ** = p<0.01

Table 9: Selected body weight data (offspring)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Mean litter body weight (PND0), g	6.453	6.464	6.711	6.454	NS
Mean litter body weight (PND4), g	10.597	10.649	9.945	9.223	NS
Mean litter body weight gain (PND0-4), g	4.142	4.157	3.419	2.760	NS
Mean litter body weight (PND13), g	32.995	33.251	30.174	28.096*	DN
Mean litter body weight gain (PND4-13), g	22.323	22.619	19.901	18.840*	DN
Mean litter body weight gain (PND0-13), g	26.542	26.784	23.587	21.575	NS

DN: Dunnett’s test, NS: Statistically not significant when compared to the control.
Statistical significance compared to control: * = p<0.05

Table 10: Anogenital distance

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Male pups
Number of evaluated litters	12	12	11	10
Anogenital distance, litter mean of males (mm)	3.43	3.45	3.40	3.20*	DU
Minimum / Maximum value, litter mean (mm)	3.1 / 3.9	3.1 / 4.0	3.2 / 3.9	3.0 / 3.4
Anogenital distance ratio by BW cube root	1.829	1.843	1.804	1.708	NS
Female pups
Number of evaluated litters	12	12	11	10
Anogenital distance, litter mean of females (mm)	1.75	1.97*	1.94	1.81	DN
Minimum / Maximum value, litter mean (mm)	1.5 / 2.1	1.5 / 2.4	1.5 / 2.3	1.7 / 2.0
Anogenital distance ratio by BW cube root	0.947	1.073	1.049	0.983	NS

NS: Statistically not significant when compared to the control. DN: Dunnett’s test, DU: Dunn’s test.
Statistical significance compared to control: * = p<0.05.

Table 11: Selected parameters related to thyroid hormone levels

Parameters	Dose groups (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000/600)
Males
Number of evaluated animals	12	12	12	12
T4 concentration (ng/mL)	62.83	61.83	61.83	61.25	NS
HC range: 52-66	difference (%)	-1.6	-1.6	-2.5
TSH concentration (ng/mL)	501.4	714.8	619.8	563.1	NS
	difference (%)	42.6	23.6	12.3
Thyroid gland weights (g)	0.0266	0.0258	0.0280	0.0245	NS
	difference (%)	-2.8	5.3	-7.8
Thyroid gland / body weight (%)	0.00578	0.00558	0.00607	0.00573	NS
	difference (%)	-3.5	5.0	-0.8
PND 13 pups
Number of evaluated litters	12	11/12#	11	10
T4 concentration (ng/mL)	61.83	60.36	57.50	52.90**	DN
HC range: 53-61	difference (%)	-2.4	-7.0	-14.4
TSH concentration (ng/mL)	729.8	904.6	1545.4	1215.2	NS
	difference (%)	24.0	111.8	66.5
Thyroid gland weights (g)	0.0060	0.0065	0.0058	0.0059	NS
	difference (%)	6.9	-4.0	-2.3
Thyroid gland / body weight (%) (x10^-4)	1.8222	1.9307	1.8752	2.2700	NS
	difference (%)	6.0	2.9	24.6

HC: Historical control.
Statistical significance compared to control: ** = p<0.01
DN: Dunnett’s test, NS: Statistically not significant when compared to the control

Conclusions:

Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were 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).

The NOAEL for reproductive effects of the parental generation: 600 mg/kg bw/day (based on no effects on fertility in the dosed groups).

The NOAEL for pups’ (F1 generation) development and survival: 600 mg/kg bw/day (based on no adverse effects on the T4 level for the PND13 pups).

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.
An apparently higher incidence of pseudopregnancy in the high dose before mating was recorded, but all mated normally with no delay in becoming sperm-positive, and mean cycle lengths were normal. Hence this was not considered to be an adverse effect of the test item (but
may have been related to the >MTD effect of treatment in the first 7 days). All pairs of animals mated, there was no effect of treatment noted during the gestation period, parturition or postpartum period in any of dose groups.
The number of implantation or liveborn pups, as well as the pre-natal and total mortality values were comparable with the control values in all test item treated dose groups. No test item related effect was observed on the number of live born pups in any of the dosed groups.
No test item-related differences were detected on the offspring body weights or body weight gains. No test item effect was observed on anogenital distance or nipple retention during the study.
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.
Test item related effect was observed in the T4 hormone level for the high dose PND 13 pups (lower values). The data were outside of the historical control range and with dose response it was considered as test item related effect but without histopathological findings and organ weight changes it was not considered as adverse. 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 and in the PND13 pups 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.

Effect on fertility: via oral route

Endpoint conclusion:

no 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.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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. An apparently higher incidence of pseudopregnancy in the high dose before mating was recorded, but all mated normally with no delay in becoming sperm-positive, and mean cycle lengths were normal. Hence this was not considered to be an adverse effect of the test item (but
may have been related to the >MTD effect of treatment in the first 7 days). All pairs of animals mated, there was no effect of treatment noted during the gestation period, parturition or postpartum period in any of dose groups.
The number of implantation or liveborn pups, as well as the pre-natal and total mortality values were comparable with the control values in all test item treated dose groups. No test item related effect was observed on the number of live born pups in any of the dosed groups.
No test item-related differences were detected on the offspring body weights or body weight gains. No test item effect was observed on anogenital distance or nipple retention during the study.
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.
Test item related effect was observed in the T4 hormone level for the high dose PND 13 pups (lower values). The data were outside of the historical control range and with dose response it was considered as test item related effect but without histopathological findings and organ weight changes it was not considered as adverse. 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 and in the PND13 pups 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 the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were 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).


• The NOAEL for reproductive effects of the parental generation: 600 mg/kg bw/day (based on no effects on fertility in the dosed groups).


• The NOAEL for pups’ (F1 generation) development and survival: 600 mg/kg bw/day (based on no adverse effects on the T4 level for the PND13 pups).

 

 

Effects on developmental toxicity

Description of key information

oral: NOAEL (male, female) maternal toxicity & developmental effects = 600 mg/kg bw/d (OECD 422; rat; oral; BASF SE 2022)

dermal: no data available

inhalative: no data available

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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:

other: 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

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

Vehicle:

water

Details on 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.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until copulation occurred, for up to 5 days
- Proof of pregnancy: the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines)
- Further matings after two unsuccessful attempts: not specified
- After successful mating each pregnant female was caged (how): single

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

Duration of test:

see "Duration of treatment / exposure"

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 (rat) dam thyroid hormones: yes
- Time of day for (rat) dam blood sampling: The timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days.

Maternal examinations:

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).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on PPD 14
- Organs examined: see table 1

OTHER:
Neurological assessment: Functional Observational Battery and locomotor activity measurement
- 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)
- Parameters: functional observation battery, including Irwin test and measurements of the landing foot splay and fore/hind grip strength; body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes (examination of uterus horns, body and cervix)
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Blood sampling:

- Plasma: Yes
- Serum: Yes
- Volume collected: not specified

Fetal examinations:

Dead pups and pups terminated on PND4 and/or PND13 were carefully examined externally for gross abnormalities
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all live rodent pups: Yes

Statistics:

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

Indices:

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

Historical control data:

Historical control data were provided by the test facility for the following parameters: body weights, food consumtion, clinical pathology testing, organ weights, Functional Observational Battery and locomotor activity measurement, delivery data, pup viability data, pup weights

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- abnormal skin colour in 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 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 females from Day 0 to Day 2. The longevity of the symptom was 3 days.
- prostration in 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 6/12 females from Day 21 to Day 49. The longevity of the symptom was 5 days.
- intermittent tremor in 7/12 females from Day 2 to Day 8. The longevity of the symptom was 1 day.
- tonic convulsion was observed in 2/12 females from Day 1 to Day 5. The longevity of the symptom was 3 days.

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 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 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:
- 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 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, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly increased cholesterol level females (by 31.4%, p<0.01); The data were within the historical control range and without histopathological relevance, it was not considered as test item related 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 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 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 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

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

not examined

Dead fetuses:

not examined

Changes in pregnancy duration:

effects observed, non-treatment-related

Description (incidence and severity):

Mid dose group:
- statistically significantly higher duration of pregnancy (p<0.05) compared to the Control, but the data were inside of the historical control range, and there was no dose-dependency

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Three non-pregnant females (one mid dose and 2 high dose animals) were observed in the study. Necropsy examination did not show any test item related change in the non-pregnant females and their male mating pairs. No microscopic changes were seen in these females or in their male pairs.

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

test mat.

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

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly lower body weight on PND13 (by -14.8%, p<0.05) and body weight gain between PND4-13 (by -15.6%, p<0.05); The data were within the historical control range therefore, it was not considered as test item related effect.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- male ADG not adjusted for body weight was slightly below the controls; It was within the historical control range and not considered as test item related effect.

Low dose group:
- statistically significantly increased AGD per litter in female pups; As it was not part of a dose response and the data were within the historical control range, it was considered as unrelated to treatment.

Changes in postnatal survival:

effects observed, non-treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly increased postnatal mortality % on PND4 (p<0.05) (within the historical control range) and thus a lower survival index at Day 4

Mid dose group:
- statistically significantly increased postnatal mortality mean and postnatal mortality % on PND4 (p<0.05) and thus a lower survival index at Day 4

Since there was no dose response and the high dose group was in the normal range, it was not considered as test item related effect.

External malformations:

no effects observed

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

High dose group:
- statistically significantly lower T4 hormone levels on PND13 (by -14.4%, p<0.01) when compared to the relevant control level; The data were outside of the historical control range and with dose response it was considered as test item related effect but without histopathological findings and organ weight changes it was not considered as adverse.

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: no adverse effects observed up to and including the highest tested dose

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Table 2: Summary of the pregnancy evaluation

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated females	12	12	11	10
Number of pregnant females	12	12	11	10
Duration of pregnancy (days)	22.25	22.33	22.82*	22.20	D
Number of implantations, mean	16.67	16.58	15.33	12.82	NS
Number of pups born, mean	14.50	14.50	15.09	12.60	NS
Number of live born pups, mean	14.50	13.58	14.45	12.20	NS
Pre-natal mortality, mean	2.17	3.00	2.27	1.90	NS
Pre-natal mortality (%), mean	12.97	17.76	13.96	15.78	NS
Post-natal mortality, mean PND0-4	0.25	0.17	3.09*	1.30	U
Post-natal mortality (%), mean PND0-4	1.53	1.90	19.02*	13.30*	U
Total mortality, mean PND4	2.42	3.17	5.36	3.20	NS
Total mortality (%), mean PND4	14.48	18.84	31.71	26.23	NS

NS: Statistically not significant when compared to the control.
D: Duncan’s multiple range test, U: Mann-Whitney U - test versus Control,
Statistical significance compared to control: * = p<0.05

Table 3: Summary of survival (offspring)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Post-natal mortality on PND0-13, mean	0.33	0.25	3.36	1.40	NS
Post-natal mortality on PND0-13 (%), mean	2.23	2.30	20.63	14.07	NS
Total mortality on PND13, mean	2.50	3.25	5.64	3.30	NS
Total mortality on PND13 (%), mean	15.00	19.39	33.26	26.94	NS
Survival index on PND0	100.00	92.11	95.37	96.11	NS
Sex ratio (%) on PND0 Female	53.33	43.93	55.91	52.16	NS
Survival index on PND4	98.47	98.25	80.98*	86.70*	U
Sex ratio (%) on PND4 Female	53.14	44.89	60.76	51.57	NS
Survival index on PND13	99.17	99.17	90.00	99.00	NS
Sex ratio (%) on PND13 Female	50.46	47.96	57.68	49.78	NS

NS: Statistically not significant when compared to the control
Sex ratio means the percentage of female pups
U: Mann-Whitney U-test versus control; Statistical significance compared to control: * = p<0.05, ** = p<0.01

Table 4: Summary of the offspring mortality

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Number of pups born	174 / 12	174 / 12	166 / 11	126/ 10	NS
Number of cannibalized pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of autolyzed pups	0 / 0	11 / 3	7 / 4	4 / 3	NA
Number of stillborn pups	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of live born pups	174 / 12	163^** / 12	159^** / 11	122^** / 10	CH
Number of found dead pups (born alive)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Number of living pups on PND0	174 / 12	163 / 12	159 / 11	122 / 10	NA
Number of cannibalized pups (PND0-13)	2 / 2	2 / 2	33 / 6	4 / 3	NA
Number of autolyzed pups (PND0-13)	2 / 2	1 / 1	4 / 2	9 / 5	NA
Number of found dead, intact pups (PND0-13)	0 / 0	0 / 0	0 / 0	0 / 0	NA
Total number of pups died (born alive)	4 / 4	3 / 3	37 / 6	13 / 6	NS
Culled for blood sampling on PND4	21 / 11	20 / 10	14 / 7	12 / 7	NA
Culled pups on PND4	30 / 9	32 / 10	13 / 4	14 / 4	NA
Number of viable pups on PND13	119 / 12	108 / 12	95 / 11	82 / 10	NS

CH: Chi Square test, NS: Statistically not significant when compared to the control,
NA: Not applicable. Statistical significance compared to control: ** = p<0.01

Table 5: Selected body weight data (offspring)

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Number of evaluated litters	12	12	11	10
Mean litter body weight (PND0), g	6.453	6.464	6.711	6.454	NS
Mean litter body weight (PND4), g	10.597	10.649	9.945	9.223	NS
Mean litter body weight gain (PND0-4), g	4.142	4.157	3.419	2.760	NS
Mean litter body weight (PND13), g	32.995	33.251	30.174	28.096*	DN
Mean litter body weight gain (PND4-13), g	22.323	22.619	19.901	18.840*	DN
Mean litter body weight gain (PND0-13), g	26.542	26.784	23.587	21.575	NS

DN: Dunnett’s test, NS: Statistically not significant when compared to the control.
Statistical significance compared to control: * = p<0.05

Table 6: Anogenital distance

Parameters	Dose group / Concentration (mg/kg bw/day)
	Control (0)	Low dose (100)	Mid dose (300)	High dose (1000/600)
Male pups
Number of evaluated litters	12	12	11	10
Anogenital distance, litter mean of males (mm)	3.43	3.45	3.40	3.20*	DU
Minimum / Maximum value, litter mean (mm)	3.1 / 3.9	3.1 / 4.0	3.2 / 3.9	3.0 / 3.4
Anogenital distance ratio by BW cube root	1.829	1.843	1.804	1.708	NS
Female pups
Number of evaluated litters	12	12	11	10
Anogenital distance, litter mean of females (mm)	1.75	1.97*	1.94	1.81	DN
Minimum / Maximum value, litter mean (mm)	1.5 / 2.1	1.5 / 2.4	1.5 / 2.3	1.7 / 2.0
Anogenital distance ratio by BW cube root	0.947	1.073	1.049	0.983	NS

NS: Statistically not significant when compared to the control. DN: Dunnett’s test, DU: Dunn’s test.
Statistical significance compared to control: * = p<0.05.

Table 7: Selected parameters related to thyroid hormone levels

Parameters	Dose groups (mg/kg bw/day)
	Control	Low (100)	Mid (300)	High (1000/600)
Males
Number of evaluated animals	12	12	12	12
T4 concentration (ng/mL)	62.83	61.83	61.83	61.25	NS
HC range: 52-66	difference (%)	-1.6	-1.6	-2.5
TSH concentration (ng/mL)	501.4	714.8	619.8	563.1	NS
	difference (%)	42.6	23.6	12.3
Thyroid gland weights (g)	0.0266	0.0258	0.0280	0.0245	NS
	difference (%)	-2.8	5.3	-7.8
Thyroid gland / body weight (%)	0.00578	0.00558	0.00607	0.00573	NS
	difference (%)	-3.5	5.0	-0.8
PND 13 pups
Number of evaluated litters	12	11/12#	11	10
T4 concentration (ng/mL)	61.83	60.36	57.50	52.90**	DN
HC range: 53-61	difference (%)	-2.4	-7.0	-14.4
TSH concentration (ng/mL)	729.8	904.6	1545.4	1215.2	NS
	difference (%)	24.0	111.8	66.5
Thyroid gland weights (g)	0.0060	0.0065	0.0058	0.0059	NS
	difference (%)	6.9	-4.0	-2.3
Thyroid gland / body weight (%) (x10^-4)	1.8222	1.9307	1.8752	2.2700	NS
	difference (%)	6.0	2.9	24.6

HC: Historical control.
Statistical significance compared to control: ** = p<0.01
DN: Dunnett’s test, NS: Statistically not significant when compared to the control

Conclusions:

Based on the existing results of this study, the following No-Observed-Adverse-Effect Levels (NOAELs) were 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).

The NOAEL for reproductive effects of the parental generation: 600 mg/kg bw/day (based on no effects on fertility in the dosed groups).

The NOAEL for pups’ (F1 generation) development and survival: 600 mg/kg bw/day (based on no adverse effects on the T4 level for the PND13 pups).

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.
An apparently higher incidence of pseudopregnancy in the high dose before mating was recorded, but all mated normally with no delay in becoming sperm-positive, and mean cycle lengths were normal. Hence this was not considered to be an adverse effect of the test item (but
may have been related to the >MTD effect of treatment in the first 7 days). All pairs of animals mated, there was no effect of treatment noted during the gestation period, parturition or postpartum period in any of dose groups.
The number of implantation or liveborn pups, as well as the pre-natal and total mortality values were comparable with the control values in all test item treated dose groups. No test item related effect was observed on the number of live born pups in any of the dosed groups.
No test item-related differences were detected on the offspring body weights or body weight gains. No test item effect was observed on anogenital distance or nipple retention during the study.
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.
Test item related effect was observed in the T4 hormone level for the high dose PND 13 pups (lower values). The data were outside of the historical control range and with dose response it was considered as test item related effect but without histopathological findings and organ weight changes it was not considered as adverse. 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 and in the PND13 pups 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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no 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.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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. No adverse effects on fertility or development were observed in a screening study in rats (OECD TG 422). As a result, the substance is not considered to be classified for fertility or developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the fifteenth time in Regulation (EC) No. 2020/1182.

During the thirteen days covered in the screening study, no effects via lactation were observed.