[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Oral: NOAEL (rat – reproductive toxicity): ≥ 1000 mg/kg bw/day, male/female, OECD TG 422, 2021

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-10-2019 to 28-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: (i) Prior to formulation: In refrigerator (2-8°C) protected from light container flushed with nitrogen ; (ii) Post-formulation: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). The dosing formulations were removed from the refrigerator and stirred at room temperature (ca. +25°C), for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Control (corn oil ; vehicle) formulations were maintained at room temperature.
- Stability under test conditions: Stable. Stability for at least 24 hours at room temperature under normal light conditions and for at least 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL in a prior conducted analytical method and formulation validation (study number cited in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation (on day 1, week 1, in all groups). Homogeneity was assessed for Groups 2 and 4 formulations by measuring the achieved concentration in the top, middle and bottom samples. Results were averaged and utilised as the concentration results. Previously conducted stability information (documented in the full study report) demonstrated that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: In a concentration range of 1 to 200 mg/mL for 8 days in the refrigerator (2-8°C) and for 24 hours at room temperature and normal laboratory conditions. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable. Substance was a liquid fully soluble in vehicle. Dosing formulations were prepared weekly as a solution, formulated in daily portions and stored in the refrigerator protected. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and continuously during the dosing procedure. Control (vehicle) formulations were maintained at room temperature.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable.
- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 422 relevant guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes. Any female without at least 2 regular estrous cycles would typically be replaced by one of the 8 spare females having at least 2 regular estrous cycles. The supernumerary females would then be removed from the study. Pre-dosing estrous cycle data were retained in the study raw data. In the present study all randomly selected females had regular estrous cycles and therefore continued within the study.
- Age at study initiation: (P) Males ca. 10 weeks (i.e. 10 – 11 weeks old) ; Females ca. 13 weeks (I.e. 13-14 weeks)
- Weight at study initiation: (P) Males: 306 – 343 g; Females: 204 – 254 g
- Fasting period before study: No.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages (Macrolon MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage housed up to 5 per cage (see above, Macrolon MIV type height 18 cm). Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles. Group housed males and females and individual housed females, including females during mating, gestation and with litters, were housed in plastic cages containing appropriate bedding and according to relevant legislative requirements. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without environmental enrichment, bedding material, food and water.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: seven days before commencement of treatment. Females: seven days between arrival and start of estrous cycle smears (females) i.e. seven days before start of the pretest period

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified pelleted diet, ad libitum – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24 (actual: 20 to 21)
- Humidity (%): 55 ± 15 (or 40 to 70 : actual 45 to 77%)
- Air changes (per hr): > 10 per hour (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2020-05-13 To: 2020-07-15

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). The dosing formulations were removed from the refrigerator and stirred at room temperature (ca. +25°C), for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Control (corn oil ; vehicle) formulations were maintained at room temperature. The stability and homogeneity of the test item formulations were determined. The formulations were determined to be acceptably stable and homogenous.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Corn oil BP was considered as appropriate based on test item solubility documented outside of the full study report (referenced within the study report).
- Concentration in vehicle: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). Test item dosing formulations were allowed to reach room temperature prior to dosing. Concentrations in vehicle were as follows: 0 (control) mg/kg/bw [or 0 mg/mL], 100 mg/kg/bw [or 20 mg/mL], 300 mg/kg/bw [or 60 mg/mL], 1000 mg/kg/bw [or 200 mg/mL]. Dose-formulations of groups 2, 3, and 4 were analysed during the study and were reported as with ± 10% applied limits.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: Information on supplier is provided in the full study report.
- Other: On post-coitum Day 19, Female #54 (100 mg/kg bw/day) was dosed at a lower volume than determined based on the body weight (1.41 mL, instead of 1.68 mL). This deviation was of a slight and incidental nature without any indications of any effects within other individuals of the relevant group. It was therefore considered not to have adversely affected the study outcome of the study.

Details on mating procedure:

-- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. Day 0 (post-coitum) [and/or Day 0 of gestation] was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles.
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability was confirmed once during the study to assess accuracy. Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation. Homogeneity was assessed for Groups 2 and 4 formulations by measuring the achieved concentration in the top, middle and bottom samples. Results were averaged and utilised as the concentration results. Previously conducted stability information (documented in the full study report) demonstrated that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: for at least 24 hours at room temperature under normal light conditions and for at least 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL in a prior conducted analytical method and formulation validation (study number cited in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation (on day 1, week 1, in all groups). Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- The analysis was performed in a validated analytical procedure. This consisted of UPLC-UV quantitative analysis with external calibration within a dedicated formulation analysis report reference within the full study report. The reference item was prepared as a Quality Control (QC) solution at 1 mg/mL (Formulation A) and 200 mg/mL (Formulation B) in the vehicle according to internal procedure. Separately, six calibration solutions in the concentration range of 20 – 120.0 mg/L were prepared from two 1000 mg/L and 2000 mg/L standard stock solutions. The end solution of the calibration solutions was tetrahydrofuran. Calibration analysis was conducted in duplicate per concentration. Study samples and QC samples were analysed by single injection. The LOQ = 1 mg/mL in vehicle. The calibration coefficient of correlation (r) = 0.992. Accuracy and precision was confirmed in the range 85 – 115% with CoV < 5%. Calibration solutions were injected throughout the validation sequence including the
beginning and end. The analytical system and/or end solutions were found to be stable if the coefficient of variation on the responses of the solutions was ≤ 20%. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- Within the study: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). Test item dosing formulations were allowed to reach room temperature prior to dosing. Concentrations in vehicle were as follows: 0 (control) mg/kg/bw [or 0 mg/mL], 100 mg/kg/bw [or 20 mg/mL], 300 mg/kg/bw [or 60 mg/mL], 1000 mg/kg/bw [or 200 mg/mL]. Dose-formulations of groups 2, 3, and 4 were analysed during the study and were reported as with ± 10% applied limits. Samples solutions were, as necessary further diluted with tetrahydrofuran to obtain concentrations within the calibration range. These were then subjected to analysis by UPLC-UV. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
- Other: Resuspension homogeneity: For a given concentration and storage condition, the deviation between the mean results of the stored formulations and the initial formulation results should be within ± 10% to be considered acceptable. For re-suspension homogeneity after storage in a refrigerator for 8 days: was demonstrated with a coefficient of variation of ≤ 5%.

Duration of treatment / exposure:

F0 Males: minimum of 28 days ; i.e. minimum of 14 days prior to mating and during the mating period and up to termination/necropsy (ca. 29 days)
F0 Females: 14 days prior to mating (with the objective to cover at least two complete estrous cycles), throughout mating, gestation and lactation (at least 13 days post-delivery) and up to the day before scheduled necropsy ; i.e. females that delivered: ca. 51 to 63 days and females that failed to deliver: 37 to 42 days.

Frequency of treatment:

Daily; at approximately the same time each day.
F1 generation were not dosed.

Details on study schedule:

- Age at study initiation: F0 Males: ca. 10 weeks (i.e. 10 – 11 weeks old) ; F0 Females: ca. 13 weeks (I.e. 13-14 weeks)
- Age at mating of the mated animals in the study: after 14 days treatment – cohabited for mating: F0 generation: Males: ca. 12 weeks ; Females: ca. 15 weeks
(i.e. after two weeks treatment)
- Age at termination: [based on ages at study initiation : [F0 Males: ca. 14 weeks old and F0 Females: that delivered ca. 21 to 23 weeks old and that failed to deliver: ca. 19 to 20 weeks old

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1; corn oil vehicle (5 mL/kg applied dose)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group 4

No. of animals per sex per dose:

10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
100 mg/kg bw/day (toxicity test) = 10
325 mg/kg bw/day (toxicity test) = 10
1000 mg/kg bw/day(toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
100 mg/kg bw/day (reproduction test) = 10
100 mg/kg bw/day (toxicity test) = 5
325 mg/kg bw/day (reproduction test) = 10
325 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (reproduction test) = 10
1000 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this study (0, 100, 300 and 1000 mg/kg bw/day) by oral gavage were chosen based upon the results obtained in a 10-day preliminary study (full details available in the full study report). Prior to this, it was concluded prior to the definitive test that the dietary route was inappropriate related to palatability issues with that route of administration. The definitive study was to be conducted by oral gavage administration. Within the 10-day range finding test: dose formulations were collected for all groups for potential analysis. Dose levels in the 10-day sighting test were: Group 1: 500 mg/kg bw/day (in corn oil), Group 2: 1000 mg/kg bw/day (in corn oil) ; both at a dose volume of 5 mL/kg with three females per dose-group. No signs of toxicity were noted at any dose level. At 500 mg/kg bw/day: No mortality, no clinical findings were seen. Slight body weight loss (up to 2%) was noted between days 1-5 of treatment, followed by body weight gain (up to 4%) between days 5-10 of treatment. Food consumption was normal. No macroscopic abnormalities were noted at necropsy. Liver and kidney weights (absolute and relative to body weight) slightly higher than normal. At 1000 mg/kg bw/day: No mortality, piloerection in 1/3 females on day 9 and 10 of treatment. Normal body weight gain was noted between days 1-5 of treatment. No body weight gain or slight body weight loss (1%) were noted between days 5-10 of treatment. Slightly reduced food consumption during days 5-10 of treatment. No macroscopic abnormalities were noted at necropsy. Liver and kidney weights (absolute and relative to body weight) slightly higher than normal. Since no clear peak effect of occurrence of clinical signs was observed in the dose range finder, clinical observations were conducted and functional observations were started in the main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- Rationale for animal assignment (if not random): Randomly assigned.
- Rationale for selecting satellite groups: Not applicable (no recovery or other satellite groups)
- Post-exposure recovery period in satellite groups: Not applicable (no recovery or other satellite groups)
- Section schedule rationale (if not random): Random

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily. Additionally, were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder with oral gavage. Detailed arena clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing. Additional functional observations were made as ‘special evaluations’. Functional performance tests were also performed on selected animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 individual body weights were recorded prior to dosing on Day 1 and at least weekly intervals thereafter. Body weights were also performed prior to termination. Full schedule: Day 1 prior to first administration and on Days 4, 8, 11, 14 and 17.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study. Except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study). Since no significant effects observed no quantitative measurements were performed.
- Time schedule for examinations: Daily (see above). Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. THYROID HORMONE ANALYSIS: Yes
- Time schedule: F0 males: after > 28 days treatment (i.e. at scheduled termination) [T4 then TSH if necessary] ; F0 females: at scheduled termination (i.e. PND 13) [T4 then TSH if necessary] or non-mated or non-pregnant F0 females: at scheduled termination [T4 then TSH if necessary]
- F1: 2 pups per litter on PND4 and/or PND14-16 [T4 where necessary]
- Assessment: T4 assessment ; TSH assessment if treatment related changes were noted. Potentially extended to T3, as appropriate.
- Note: Measurement of total T4 was conducted for F0-males and PND 14-16 pups. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16. Serum samples retained for possible future analysis were maintained by the testing facility in the freezer (≤-75°C).

Oestrous cyclicity (parental animals):

ESTROUS CYCLE: Yes
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage (vaginal smears)
- Daily performed for all F0 females beginning 14 days prior to treatment (pretest), first 14 days of treatment, during mating until evidence of copulation/mating or separation from the male.
- Daily performance for those females with no evidence of copulation until termination of mating period.
- Final vaginal lavage taken on day of necropsy (except for females that indicated spontaneous mortality or when humanely terminate in extremis).

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations:
testis weight, epididymis weight and histology/histopathology

For the testes of all males of Groups 1 and 4 (along with selected male #11 and #18 in group 2), a detailed qualitative histopathology examination was made, where appropriate taking into account the tubular stages of the spermatogenic cycle. Specifically, testes were evaluated to assess the progression of stages of the spermatogenic cycle, cell associations, and proportions expected to be present during spermatogenesis along with assessment of interstitial and supporting cell types (Leydig cells, macrophages, vasculature, and rete testis). Any cell- or stage-specificity of testicular findings were noted. The epididymis was also subject to histology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: clinical observations, litter size, sex ratio, survival indices, ano-genital distance, body weight change, presence of nipple/areolae count in male offspring , macroscopic pathology / abnormalities. Histopathology. Thyroid Hormone Analysis.

GROSS EXAMINATION OF DEAD PUPS:
Yes, where required or if applicable.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: F0 Males: After 4 weeks treatment (ca. 29 days)
- Maternal animals: F0 females:14 days prior to mating (with the objective to cover at least two complete estrous cycles), throughout mating, gestation and lactation (at least 13 days post-delivery) and up to the day before scheduled necropsy ; i.e. females that delivered: ca. 51 to 63 days and females that failed to deliver: 37 to 42 days.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Cranial cavity and all orifices. With special attention to reproductive organs.
- organs weighed: Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues were prepared for microscopic examination and weighed, respectively. For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed at scheduled intervals.
- Organs and tissues preserved in neutral buffered 10% formalin: Identification marks: not processed, Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain, [cerebellum, mid-brain, cortex], (Pituitary gland), Caecum, (Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides *, Seminal vesicles including coagulating gland, Eyes (including optic nerve [if detectable] and harderian gland) *, Skeletal muscle (Skin), (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, Femur including joint, Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes *, Kidneys, Thymus (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, (Oesophagus), All gross lesions
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from selected animals and unscheduled mortality or terminated in extremis : animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, larynx, optic nerve, pancreas, skin and tongue
- all gross lesions
- Males that failed to sire (except for males which were selected) and females that failed to deliver pups : Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- For the testes of all selected males of Groups 1 and 4, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at PND 14-16 days of age. Except for the two pups per litter selected for blood collection which were sacrificed PND 14-16 separately.
- Fetuses of females that were euthanized in extremis were subject to examination and sexed.
- On PND 4, the surplus pups were euthanized. From two surplus pups per litter, blood was collected, if possible.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Particular attention was paid to the external genitalia. Thyroids were retained from one male and one female offspring in each litter at PND 14-16 of age (see blood collection, above)

HISTOPATHOLOGY / ORGAN WEIGHTS
The thyroid tissues were prepared for microscopic examination from one male and one female offspring in each litter. Organ weights were not performed for F1 males or F1 females.

Statistics:

Refer to 'Any other information on materials and methods incl. tables' for detailed information on statistics.

Reproductive indices:

Mating index (%) or Copulation index (%) ; Precoital time interval ; Fertility index (%) ; Gestation index (%) ; Duration of gestation ; Number of Implantation Sites

Offspring viability indices:

Parturition/Maternal Care ; Pre-birth loss (%) ; Viability index (%) ; Post-implantation survival index (%) ; Live Birth index (%) ; Lactation Index (%) ; litter size and Sex Ratio

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.

Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups from Day 13 of dosing onwards until the end of the treatment period, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect, and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment related mortalities.

One female (#78) treated at 1000 mg/kg was humanely terminated (in extremis) on day 38 (during delivery). Due to difficulties during delivery (vaginal blood loss for 2 days with no pups delivered). Pregnancy for this female was confirmed based on palpation (no evidence of mating). Macroscopic observation revealed discolouration of the thyroid and a thymus reduced in size. Six implantation sites were noted and the uterus contained six (6) live fetuses.

Mortality was considered to be unrelated to treatment with the test item due to the incidental occurrence.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects on group mean body weight change and/or body weight gain, in males/females up to 1000 mg/kg bw/day. weights and body weight gain were observed in males and females up to 1000 mg/kg bw/day. Any statistically significant changes in body weights (lactation Days 1 and 7 at 300 mg/kg bw/day) and body weight gain (lactation Day 13 at all dose levels) were considered to be unrelated to treatment with the test item. This was due to an absence of dose-response.

For females in gestation and lactation:
There were no test item related effects on group mean body weight change during gestation and lactation in reproductive phase females. The occasional statistical significances noted at 300 mg/kg bw/day were considered to reflect normal variation rather than an effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant test item related effects on food consumption of males or females over Weeks 1 to 6 or for females during gestation and lactation.

One female (#76) at 1000 mg/kg bw/day) had a relative food consumption that was relatively high over Days 11-14 post-coitum and low over Days 14-17 post-coitum. Due to the single occurrence, this was considered to be unrelated to treatment.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

Description (incidence and severity):

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was conducted as no effect was suspected.

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no reported effects (with dose-response and/or statistical significance) to the eyes (in life or post termination) in the parameters examined.

Exophthalmus, was seen in one female (#68) at 300 mg/kg bw/day only post termination.

Haematological findings:

no effects observed

Description (incidence and severity):

Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects.

All inter-group differences from controls to treatment groups at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Specifically, the statistically significantly decreased red blood cell distribution width in males treated at 100 mg/kg bw/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant higher level of alkaline phosphatase (ALP) was observed in females treated at 1000 mg/kg bw/day (x2.6 of control; mean value above the historical control data range), which also tended to be increased in males at 1000 mg/kg bw/day (but without achieving statistical significance).

The statistically significant lower level of total bilirubin in males treated at 1000 mg/kg bw/day (x0.8 of control), was considered not toxicologically relevant, since 3 of 5 individual values were within and 2 of 5 individual values were only marginally below the concurrent control group range. 2 of 5 values of the concurrent control group were at the upper limit of the historical range, and the mean was within the historical control data range.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength, Motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related gross pathological/macroscopic abnormalities detected.

Within histopathology:
An increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys of 5 of 5 males at 1000 mg/kg bw/day (minimal-to slight). The incidences recorded in the remaining dose groups including controls were: control group: 2 of 5 males (minimal to slight) ; 300 mg/kg bw/day: 1 of 5 minimal and/or were within background.

Female livers showed a variable amount of cytoplasmic rarefaction (most likely glycogen storage) which did not show dose-response and was considered to be related to the non-fasted state of these females and therefore unrelated to the treatment. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Test item-related histopathological alterations present in the kidneys of males treated at 1000 mg/kg bw/day consisted of an increased incidence and severity in hyaline droplet accumulation. The hyaline droplet accumulation recorded in the male kidneys was considered to relate to alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in mammals, including man. Additionally, the minimal to slight degree severities, did not exceed the severities recorded for control males of the current study, and the hyaline droplet accumulation was not accompanied by indicators of tubular damage. This alteration was therefore regarded as non-adverse.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathological/macroscopic abnormalities detected. There were no microscopic neoplastic findings that were considered to be related to treatment with the test item.

Other effects:

no effects observed

Description (incidence and severity):

1. Thyroid Hormone Assessment:
Serum levels of T4 in F0-males were statistically significantly decreased in F0-males and F0 females at 300 and 1000 mg/kg bw/day (males: x0.8 and x0.8 of control, respectively and females: x0.8 and x0.7 of control, respectively). Mean T4 values remained within the historical control data range.

F0 generation :
Males Day 30 :
Control : T4 (ng/mL): mean (n=10) = 48.69 (SD: 6.26)
100 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 48.69 (SD: 7.86)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 40.47 (SD: 5.19)
1000 mg/kg bw/day: T4 (ng/mL): mean (n-10) = 40.04 (SD: 8.03)
Females Day 51 :
Control : T4 (ng/mL): mean (n=9) = 32.99 (SD: 9.61)
100 mg/kg bw/day: T4 (ng/mL): mean (n=8) = 28.38 (SD: 5.52)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 24.83 (SD: 4.66)
1000 mg/kg bw/day: T4 (ng/mL): mean (n=9) = 21.71 (SD: 2.49)

Historical control data for Wistar Han rats (period 2017 to 2020) - presented in the full study report :
F0-males: T4 (ng/mL): mean = 46.5 ; P5 – P95 = 31.30-65.20 (n=430).
F0 females: T4 (ng/mL): mean = 27.8 ; P5 – P95 = 16.70-40.40 (n=190).

Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item.

Additionally, F1 generation serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item related effects on estrous cycle regularity during the 2 weeks prior to pairing. All females showed regular 4-5 day cycles, with the exception of one female at 100 mg/kg bw/day (#53) and one female at 1000 mg/kg bw/day (#75). Both females had normal litters, had an extended di-estrous and were unable to be classified. Given their incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, these findings did not indicate a relation with treatment

There was no effect of treatment on gestation length, with all gestation lengths within the expected range of 22 to 23 days. The gestation indices were respectively: 100%, 89%, 100% and 90% for the control, 100, 300 and 1000 mg/kg bw/day groups. Failed delivery of females (#58 at 100 mg/kg bw/day and #78 humanely terminated, at 1000 mg/kg bw/day during delivery) was without related histopathology findings in reproductive organs and was considered unrelated to treatment.

The number of implantations (implantation sites) was considered unaffected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no treatment related findings. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

The apparent : statistically significant higher testes weight (relative to body weight only) in males at 300 and 1000 mg/kg bw/day was considered to be the result of a potential (non-adverse) test item-related effect on final body weight. Rather than an adverse effect on the testes related to treatment. Additionally, there was a lack of a dose-response in terms of organ/body weight ratio (%) and pathological and histopathological correlates. Furthermore, stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was not affect on fertility or mating performance related to treatment.

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No toxicologically significant clinical signs occurred among offspring (F1) that were considered to be related to treatment with the test item.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no treatment related mortality associated with treatment within F1 males or females. This is considering that there was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, absence of adverse effects to nipple counts and/or macropathology findings in offspring.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 100% for all groups. No signs of difficult or prolonged parturition were noted among the pregnant females, except for Female #78 at 1000 mg/kg bw/day. This female was humanely terminated (in extremis) due to delivery problems. Six live fetuses were noted in the uterus and this was not considered a test item related finding.

The number of implantations (implantation sites) was considered unaffected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study).

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 100%, 96%, 100% and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Three pups at 100 mg/kg bw/day from the same litter (female #60) were missing on PND 2. Missing offspring were most likely cannibalised. No toxicological relevance was attributed to these findings since the mortality incidence did not show a dose-related trend and within the expected range considered normal for pups of this age.

In addition, there were no toxicologically relevant macroscopic/pathological findings observed for offspring.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no treatment related effect on body weight associated with treatment within F1 males or females.

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

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant macroscopic/pathological findings observed for offspring.

Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. The statistically significantly higher mean for normalized anogenital distance of male pups at 300 mg/kg bw/day occurred without a dose response. This variation was considered not to be related to treatment.

Histopathological findings:

no effects observed

Description (incidence and severity):

There were no other macroscopic findings or needed histopathological finding observed that were attributable to parental treatment with the test item.

See 'Other Effects' for Thyroid Hormone Analysis and/or any related Histopathology (as applicable)

Other effects:

no effects observed

Description (incidence and severity):

1. Thyroid Hormone Assessment:
Serum levels of T4 in F0-males were statistically significantly decreased in F0-males and F0 females at 300 and 1000 mg/kg bw/day (males: x0.8 and x0.8 of control, respectively and females: x0.8 and x0.7 of control, respectively). Mean T4 values remained within the historical control data range.

F0 generation :
Males Day 30 :
Control : T4 (ng/mL): mean (n=10) = 48.69 (SD: 6.26)
100 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 48.69 (SD: 7.86)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 40.47 (SD: 5.19)
1000 mg/kg bw/day: T4 (ng/mL): mean (n-10) = 40.04 (SD: 8.03)
Females Day 51 :
Control : T4 (ng/mL): mean (n=9) = 32.99 (SD: 9.61)
100 mg/kg bw/day: T4 (ng/mL): mean (n=8) = 28.38 (SD: 5.52)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 24.83 (SD: 4.66)
1000 mg/kg bw/day: T4 (ng/mL): mean (n=9) = 21.71 (SD: 2.49)

Historical control data for Wistar Han rats (period 2017 to 2020) - presented in the full study report :
F0-males: T4 (ng/mL): mean = 46.5 ; P5 – P95 = 31.30-65.20 (n=430).
F0 females: T4 (ng/mL): mean = 27.8 ; P5 – P95 = 16.70-40.40 (n=190).

Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item.

F1 offspring:
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

There were no other macroscopic findings finding observed in the offspring that were attributable to parental treatment with the test item.

2. Offspring Ano-Genital Distance and Male Nipple Counts
Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. The statistically significantly higher mean for normalized anogenital distance of male pups at 300 mg/kg bw/day occurred without a dose response. This variation was considered not to be related

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study.

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index (relationship to treatment could not be excluded and therefore on a precautionary basis : reflected in the conclusion of the present screening study).

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 10-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by oral gavage administration for at least four weeks. Three treatment groups with a control were conducted, each comprising ten male and ten female rats which received oral gavage test item at doses of 0 (Control), 100, 300, 1000 mg/kg bw/day test item in corn oil vehicle.Chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity.Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 37-42 days. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received the vehicle, corn oil, at the same dose volume as treated groups.The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development: mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups). No treatment-related changes were noted in any of the following parameters investigated in this study, i.e. clinical appearance, functional observations, motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex, body weight, food consumption, clinical laboratory investigations (haematology, clotting parameters), and macroscopic examination. One female (no. 78) treated at 1000 mg/kg was humanely terminated (in extremis) on day 38 (during delivery). Due to difficulties during delivery (vaginal blood loss for 2 days with no pups delivered). Pregnancy for this female was confirmed based on palpation (no evidence of mating). Macroscopic observation revealed discolouration of the thyroid and a thymus reduced in size. Six implantation sites were noted, and the uterus contained six (6) live fetuses. Mortality was considered to be unrelated to treatment with the test item due to the incidental occurrence.

Test item-related morphologic alterations were present in the kidneys of males treated at 1000 mg/kg bw/day and consisted of an increased incidence and severity in hyaline droplet accumulation. The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha-2u-globulin. This male rat specific protein is not present in female rats nor in mammals, including man (ref: P.S. Sahota et al., 2013). The severities recorded at 1000 mg/kg bw/day (up to slight degree) did not exceed the severities recorded for control males of the current study, and the hyaline droplet accumulation was not accompanied by indicators of tubular damage and was therefore considered a non-adverse finding. Test item-related higher liver weights (absolute and relative to body weight) in males at 1000 mg/kg bw/day and increased alkaline phosphatase (ALP) in males (not achieving statistical significance) and females was recorded. Higher (absolute and relative to body weight) kidney weights were recorded in males at 300 and 1000 mg/kg bw/day. These effects observed in liver and kidney occurred without macroscopic and microscopic correlates and were therefore considered a non-adverse finding.

In males and females at 300 and 1000 mg/kg/day a test item related decrease in total T4 levels was recorded (in males 80% and 80% of control, respectively and in females 80% and 70% of control, respectively) . Although mean values remained within the historical control data range. Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item. Within reproductive parameters, no toxicologically significant changes were noted in this study, i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs.

In developmental parameters, a lower post implantation survival index was recorded at 1000 mg/kg bw/day (93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively). These changes could only partly be attributed to the female (no. 78) at 1000 mg/kg bw/day that was humanely sacrificed due to delivery difficulties. Even if the pups of this female were born, the post-implantation survival index would still be lower compared to the concurrent control. Therefore, a possible relation with treatment could not be excluded. As a consequence of the lower post-implantation survival index, litter size was also decreased in females treated at 1000 mg/kg bw/day. No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

 

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/developmental screening study – Oral:

Key study : OECD TG 422, 2021 : The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 10-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by oral gavage administration for at least four weeks. Three treatment groups with a control were conducted, each comprising ten male and ten female rats which received oral gavage test item at doses of 0d histopathological examination of reproductive organs. In developmental parameters, a lower post implantation survival index was recorded at 1000 mg/kg bw/day. These changes could only partly be attributed to the female (no. 78) at 1000 mg/kg bw/day that was humanely sacrificed due to delivery difficulties. Even if the pups of this female were born, the post-implantation survival index would still be lower compared to the concurrent control. Therefore, a possible relation with treatment could not be excluded. As a consequence of the lower post-implantation survival index, litter size was also decreased in females treated at 1000 mg/kg bw/day. No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination. Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index.

Effects on developmental toxicity

Description of key information

Oral: NOAEL (rat – developmental toxicity): ≥ 300 mg/kg bw/day based on apparent decreased post-implantation survival index, male/female, OECD TG 422, 2021

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25-10-2019 to 28-10-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: Not applicable.
- Specific activity: Not applicable.
- Locations of the label: Not applicable.
- Expiration date of radiochemical substance: Not applicable.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: (i) Prior to formulation: In refrigerator (2-8°C) protected from light container flushed with nitrogen ; (ii) Post-formulation: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). The dosing formulations were removed from the refrigerator and stirred at room temperature (ca. +25°C), for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Control (corn oil ; vehicle) formulations were maintained at room temperature.
- Stability under test conditions: Stable. Stability for at least 24 hours at room temperature under normal light conditions and for at least 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL in a prior conducted analytical method and formulation validation (study number cited in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation (on day 1, week 1, in all groups). Homogeneity was assessed for Groups 2 and 4 formulations by measuring the achieved concentration in the top, middle and bottom samples. Results were averaged and utilised as the concentration results. Previously conducted stability information (documented in the full study report) demonstrated that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: In a concentration range of 1 to 200 mg/mL for 8 days in the refrigerator (2-8°C) and for 24 hours at room temperature and normal laboratory conditions. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Not applicable. Substance was a liquid fully soluble in vehicle. Dosing formulations were prepared weekly as a solution, formulated in daily portions and stored in the refrigerator protected. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and continuously during the dosing procedure. Control (vehicle) formulations were maintained at room temperature.
- Preliminary purification step (if any): Not applicable.
- Final dilution of a dissolved solid, stock liquid or gel: Not applicable.
- Final preparation of a solid: Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material): Liquid formulations in vehicle.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable): Not applicable.

OTHER SPECIFICS: Not applicable.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Females (if applicable) nulliparous and non-pregnant: Yes. Any female without at least 2 regular estrous cycles would typically be replaced by one of the 8 spare females having at least 2 regular estrous cycles. The supernumerary females would then be removed from the study. Pre-dosing estrous cycle data were retained in the study raw data. In the present study all randomly selected females had regular estrous cycles and therefore continued within the study.
- Age at study initiation: (P) Males ca. 10 weeks (i.e. 10 – 11 weeks old) ; Females ca. 13 weeks (I.e. 13-14 weeks)
- Weight at study initiation: (P) Males: 306 – 343 g; Females: 204 – 254 g
- Fasting period before study: No.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages (Macrolon MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage housed up to 5 per cage (see above, Macrolon MIV type height 18 cm). Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles. Group housed males and females and individual housed females, including females during mating, gestation and with litters, were housed in plastic cages containing appropriate bedding and according to relevant legislative requirements. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without environmental enrichment, bedding material, food and water.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified pelleted diet, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: seven days before commencement of treatment. Females: seven days between arrival and start of estrous cycle smears (females) i.e. seven days before start of the pretest period

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified pelleted diet, ad libitum – batch numbers and certificates of analysis provided in the full study report. The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24 (actual: 20 to 21)
- Humidity (%): 55 ± 15 (or 40 to 70 : actual 45 to 77%)
- Air changes (per hr): > 10 per hour (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2020-05-13 To: 2020-07-15

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). The dosing formulations were removed from the refrigerator and stirred at room temperature (ca. +25°C), for at least 30 minutes before dosing and dosed within 6 hours after removal from the refrigerator. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Control (corn oil ; vehicle) formulations were maintained at room temperature. The stability and homogeneity of the test item formulations were determined. The formulations were determined to be acceptably stable and homogenous.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Corn oil BP was considered as appropriate based on test item solubility documented outside of the full study report (referenced within the study report).
- Concentration in vehicle: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). Test item dosing formulations were allowed to reach room temperature prior to dosing. Concentrations in vehicle were as follows: 0 (control) mg/kg/bw [or 0 mg/mL], 100 mg/kg/bw [or 20 mg/mL], 300 mg/kg/bw [or 60 mg/mL], 1000 mg/kg/bw [or 200 mg/mL]. Dose-formulations of groups 2, 3, and 4 were analysed during the study and were reported as with ± 10% applied limits.
- Amount of vehicle (if gavage): Treatment volume was 5 mL/kg for control (negative, untreated group) and all treatment groups with applicable test item concentrations per group.
- Purity: Information on supplier is provided in the full study report.
- Other: On post-coitum Day 19, Female #54 (100 mg/kg bw/day) was dosed at a lower volume than determined based on the body weight (1.41 mL, instead of 1.68 mL). This deviation was of a slight and incidental nature without any indications of any effects within other individuals of the relevant group. It was therefore considered not to have adversely affected the study outcome of the study.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The homogeneity and stability was confirmed once during the study to assess accuracy. Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation. Homogeneity was assessed for Groups 2 and 4 formulations by measuring the achieved concentration in the top, middle and bottom samples. Results were averaged and utilised as the concentration results. Previously conducted stability information (documented in the full study report) demonstrated that the test item formulation is stable when prepared and stored under the same conditions as those used in the present study, as follows: for at least 24 hours at room temperature under normal light conditions and for at least 8 days in the refrigerator was confirmed over the concentration range 1 to 200 mg/mL in a prior conducted analytical method and formulation validation (study number cited in the full study report).
- Solubility and stability of the test substance in the solvent/vehicle: Formulations were analysed for the assessment of achieved concentration of test item in formulations and verification of the absence of test item in the control formulation (on day 1, week 1, in all groups). Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- The analysis was performed in a validated analytical procedure. This consisted of UPLC-UV quantitative analysis with external calibration within a dedicated formulation analysis report reference within the full study report. The reference item was prepared as a Quality Control (QC) solution at 1 mg/mL (Formulation A) and 200 mg/mL (Formulation B) in the vehicle according to internal procedure. Separately, six calibration solutions in the concentration range of 20 – 120.0 mg/L were prepared from two 1000 mg/L and 2000 mg/L standard stock solutions. The end solution of the calibration solutions was tetrahydrofuran. Calibration analysis was conducted in duplicate per concentration. Study samples and QC samples were analysed by single injection. The LOQ = 1 mg/mL in vehicle. The calibration coefficient of correlation (r) = 0.992. Accuracy and precision was confirmed in the range 85 – 115% with CoV < 5%. Calibration solutions were injected throughout the validation sequence including the
beginning and end. The analytical system and/or end solutions were found to be stable if the coefficient of variation on the responses of the solutions was ≤ 20%. Stability results were considered acceptable if the sample analysis results were within or equal to ±10% of the concentration determined by the initial analysis of each formulation and ±10% of the exact or calculated concentration. Analysis of formulations after storage yielded a relative difference of ≤ 10%. The test item was considered stable in vehicle formulations during storage.
- Within the study: The test item was prepared as a solution in the vehicle at concentrations of 20, 60 and 200 mg/mL. The dosing formulations were prepared weekly as a clear solution filled out in daily portions and stored in the refrigerator (+4°C). Test item dosing formulations were allowed to reach room temperature prior to dosing. Concentrations in vehicle were as follows: 0 (control) mg/kg/bw [or 0 mg/mL], 100 mg/kg/bw [or 20 mg/mL], 300 mg/kg/bw [or 60 mg/mL], 1000 mg/kg/bw [or 200 mg/mL]. Dose-formulations of groups 2, 3, and 4 were analysed during the study and were reported as with ± 10% applied limits. Samples solutions were, as necessary further diluted with tetrahydrofuran to obtain concentrations within the calibration range. These were then subjected to analysis by UPLC-UV. The analytical method was validated (details available within the full study report).
- Mean concentrations of dose-formulations analysed during the study were within ± 10% applied limits confirming accurate test item/vehicle formulation.
- Other: Resuspension homogeneity: For a given concentration and storage condition, the deviation between the mean results of the stored formulations and the initial formulation results should be within ± 10% to be considered acceptable. For re-suspension homogeneity after storage in a refrigerator for 8 days: was demonstrated with a coefficient of variation of ≤ 5%.

Details on mating procedure:

- M/F ratio per cage: 1:1 within the same treatment groups.
- Length of cohabitation: up to 2 weeks
- Proof of pregnancy: evidence of sperm in the vaginal lavage and/or by the appearance of an intravaginal copulatory plug. Day 0 (post-coitum) [and/or Day 0 of gestation] was when positive evidence of mating was detected (daily checks).
- Further matings after unsuccessful attempts: No.
- After successful mating each pregnant female was caged (how): Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles.
- Any other deviations from standard protocol: No

Duration of treatment / exposure:

F0 Males: minimum of 28 days ; i.e. minimum of 14 days prior to mating and during the mating period and up to termination/necropsy (ca. 29 days)
F0 Females: 14 days prior to mating (with the objective to cover at least two complete estrous cycles), throughout mating, gestation and lactation (at least 13 days post-delivery) and up to the day before scheduled necropsy ; i.e. females that delivered: ca. 51 to 63 days and females that failed to deliver: 37 to 42 days.

Frequency of treatment:

Daily; at approximately the same time each day.
F1 generation were not dosed.

Duration of test:

- Age at study initiation: F0 Males: ca. 10 weeks (i.e. 10 – 11 weeks old) ; F0 Females: ca. 13 weeks (I.e. 13-14 weeks)
- Age at mating of the mated animals in the study: after 14 days treatment – cohabited for mating: F0 generation: Males: ca. 12 weeks ; Females: ca. 15 weeks
(i.e. after two weeks treatment)
- Age at termination: [based on ages at study initiation : [F0 Males: ca. 14 weeks old and F0 Females: that delivered ca. 21 to 23 weeks old and that failed to deliver: ca. 19 to 20 weeks old

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control – Group 1; corn oil vehicle (5 mL/kg applied dose)

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High – Group 4

No. of animals per sex per dose:

10 per sex per dose (10 male / 10 female)
F1 generation were not dosed.

Males:
Control (toxicity test) = 5
Control (recovery phase) = 5
100 mg/kg bw/day (toxicity test) = 10
325 mg/kg bw/day (toxicity test) = 10
1000 mg/kg bw/day(toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Females:
Control (reproduction test) = 10
Control (toxicity test) = 5
Control (recovery period) = 5
100 mg/kg bw/day (reproduction test) = 10
100 mg/kg bw/day (toxicity test) = 5
325 mg/kg bw/day (reproduction test) = 10
325 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (reproduction test) = 10
1000 mg/kg bw/day (toxicity test) = 5
1000 mg/kg bw/day (recovery period) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this study (0, 100, 300 and 1000 mg/kg bw/day) by oral gavage were chosen based upon the results obtained in a 10-day preliminary study (full details available in the full study report). Prior to this, it was concluded prior to the definitive test that the dietary route was inappropriate related to palatability issues with that route of administration. The definitive study was to be conducted by oral gavage administration. Within the 10-day range finding test: dose formulations were collected for all groups for potential analysis. Dose levels in the 10-day sighting test were: Group 1: 500 mg/kg bw/day (in corn oil), Group 2: 1000 mg/kg bw/day (in corn oil) ; both at a dose volume of 5 mL/kg with three females per dose-group. No signs of toxicity were noted at any dose level. At 500 mg/kg bw/day: No mortality, no clinical findings were seen. Slight body weight loss (up to 2%) was noted between days 1-5 of treatment, followed by body weight gain (up to 4%) between days 5-10 of treatment. Food consumption was normal. No macroscopic abnormalities were noted at necropsy. Liver and kidney weights (absolute and relative to body weight) slightly higher than normal. At 1000 mg/kg bw/day: No mortality, piloerection in 1/3 females on day 9 and 10 of treatment. Normal body weight gain was noted between days 1-5 of treatment. No body weight gain or slight body weight loss (1%) were noted between days 5-10 of treatment. Slightly reduced food consumption during days 5-10 of treatment. No macroscopic abnormalities were noted at necropsy. Liver and kidney weights (absolute and relative to body weight) slightly higher than normal. Since no clear peak effect of occurrence of clinical signs was observed in the dose range finder, clinical observations were conducted and functional observations were started in the main study after dosing at no specific time point, but within a similar time period after dosing for the respective animals.
- Rationale for animal assignment (if not random): Randomly assigned.
- Rationale for selecting satellite groups: Not applicable (no recovery or other satellite groups)
- Post-exposure recovery period in satellite groups: Not applicable (no recovery or other satellite groups)
- Section schedule rationale (if not random): Random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily. Additionally, were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals. During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder with oral gavage. Detailed arena clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment. These observations were conducted after dosing. Additional functional observations were made as ‘special evaluations’. Functional performance tests were also performed on selected animals during Week 4, together with an assessment of sensory reactivity to different stimuli.

BODY WEIGHT: Yes
- Time schedule for examinations: F0 individual body weights were recorded prior to dosing on Day 1 and at least weekly intervals thereafter. Body weights were also performed prior to termination. Full schedule: Day 1 prior to first administration and on Days 4, 8, 11, 14 and 17.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not applicable.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable.
- Other: Food consumption was recorded for each cage group at weekly intervals throughout the study. Except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes but only visual observation (not drinking water study). Since no significant effects observed no quantitative measurements were performed.
- Time schedule for examinations: Daily (see above). Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected. Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles.

OTHER:
1, See ‘FOOD CONSUMPTION’ field.
2. THYROID HORMONE ANALYSIS: Yes
- Time schedule: F0 males: after > 28 days treatment (i.e. at scheduled termination) [T4 then TSH if necessary] ; F0 females: at scheduled termination (i.e. PND 13) [T4 then TSH if necessary] or non-mated or non-pregnant F0 females: at scheduled termination [T4 then TSH if necessary]
- F1: 2 pups per litter on PND4 and/or PND14-16 [T4 where necessary]
- Assessment: T4 assessment ; TSH assessment if treatment related changes were noted. Potentially extended to T3, as appropriate.
- Note: Measurement of total T4 was conducted for F0-males and PND 14-16 pups. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 14-16 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 14-16. Serum samples retained for possible future analysis were maintained by the testing facility in the freezer (≤-75°C).

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes (atlthough noting the requirement for corpora lutea counts is also no longer included in the OECD422 guideline. Therefore this observation has no impact on the regulatory acceptability/compliance of the study).
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
- Other: The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]

Statistics:

Refer to 'Any other information on materials and methods incl. tables' for detailed information on statistics.

Indices:

Mating index (%) or Copulation index (%) ; Precoital time interval ; Fertility index (%) ; Gestation index (%) ; Duration of gestation ; Number of Implantation Sites ; Parturition/Maternal Care ; Pre-birth loss (%) ; Viability index (%) ; Live Birth index (%) ; Lactation Index (%) ; litter size and Sex Ratio

Historical control data:

Historical control data was available.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.

Salivation seen after dosing among animals of the 300 and 1000 mg/kg bw/day dose groups from Day 13 of dosing onwards until the end of the treatment period, was considered not toxicologically relevant, taking into account the nature and minor severity of the effect, and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female (#78) treated at 1000 mg/kg was humanely terminated (in extremis) on day 38 (during delivery). Due to difficulties during delivery (vaginal blood loss for 2 days with no pups delivered). Pregnancy for this female was confirmed based on palpation (no evidence of mating). Macroscopic observation revealed discolouration of the thyroid and a thymus reduced in size. Six implantation sites were noted and the uterus contained six (6) live fetuses.

Mortality was considered to be unrelated to treatment with the test item due to the incidental occurrence.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related effects on group mean body weight change and/or body weight gain, in males/females up to 1000 mg/kg bw/day. weights and body weight gain were observed in males and females up to 1000 mg/kg bw/day. Any statistically significant changes in body weights (lactation Days 1 and 7 at 300 mg/kg bw/day) and body weight gain (lactation Day 13 at all dose levels) were considered to be unrelated to treatment with the test item. This was due to an absence of dose-response.

For females in gestation and lactation:
There were no test item related effects on group mean body weight change during gestation and lactation in reproductive phase females. The occasional statistical significances noted at 300 mg/kg bw/day were considered to reflect normal variation rather than an effect of treatment.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no statistically significant test item related effects on food consumption of males or females over Weeks 1 to 6 or for females during gestation and lactation.

One female (#76) at 1000 mg/kg bw/day) had a relative food consumption that was relatively high over Days 11-14 post-coitum and low over Days 14-17 post-coitum. Due to the single occurrence, this was considered to be unrelated to treatment.

Food efficiency:

not examined

Description (incidence and severity):

See 'Food consumption'

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

no effects observed

Description (incidence and severity):

Water consumption was monitored on regular basis throughout the study by visual inspection of the water bottles. No quantitative investigation was conducted as no effect was suspected.

Ophthalmological findings:

not examined

Description (incidence and severity):

There were no reported effects (with dose-response and/or statistical significance) to the eyes (in life or post termination) in the parameters examined.

Exophthalmus, was seen in one female (#68) at 300 mg/kg bw/day only post termination.

Haematological findings:

no effects observed

Description (incidence and severity):

Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects.

All inter-group differences from controls to treatment groups at the end of treatment were minor in nature, lacked dose-relationship or were confined to one sex; these were consequently attributed to normal biological variation.

Specifically, the statistically significantly decreased red blood cell distribution width in males treated at 100 mg/kg bw/day was considered to be unrelated to treatment as this occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant higher level of alkaline phosphatase (ALP) was observed in females treated at 1000 mg/kg bw/day (x2.6 of control; mean value above the historical control data range), which also tended to be increased in males at 1000 mg/kg bw/day (but without achieving statistical significance).

The statistically significant lower level of total bilirubin in males treated at 1000 mg/kg bw/day (x0.8 of control), was considered not toxicologically relevant, since 3 of 5 individual values were within and 2 of 5 individual values were only marginally below the concurrent control group range. 2 of 5 values of the concurrent control group were at the upper limit of the historical range, and the mean was within the historical control data range.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory reactivity and grip strength, Motor activity scores and arena observations appeared normal and were considered unaffected by treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A higher liver weight (absolute and relative to body weight) was recorded for males at 1000 mg/kg bw/day. There was no microscopic correlate for this weight change.

Remaining differences that reached statistical significance, included higher thyroid gland weight (absolute and relative to body weight) and higher adrenal gland weight (relative to body weight only) in males at 300 mg/kg bw/day and higher kidney weights (relative to body weight only) in females at 300 mg/kg bw/day, were considered not to be test item-related due to the lack of dose-response and/or histopathological/microscopic correlates.

The apparent : statistically significant higher testes weight (relative to body weight only) in males at 300 and 1000 mg/kg bw/day was considered to be the result of a potential (non-adverse) test item-related effect on final body weight. Rather than an adverse effect on the testes related to treatment. Additionally, there was a lack of a dose-response in terms of organ/body weight ratio (%) and pathological and histopathological correlates. Furthermore, stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item-related gross pathological findings.

The incidence and distribution of all findings were considered incidental and unrelated to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related gross pathological/macroscopic abnormalities detected.

Within histopathology:
An increased incidence and severity of hyaline droplet accumulation was recorded in the kidneys of 5 of 5 males at 1000 mg/kg bw/day (minimal-to slight). The incidences recorded in the remaining dose groups including controls were: control group: 2 of 5 males (minimal to slight) ; 300 mg/kg bw/day: 1 of 5 minimal and/or were within background.

Female livers showed a variable amount of cytoplasmic rarefaction (most likely glycogen storage) which did not show dose-response and was considered to be related to the non-fasted state of these females and therefore unrelated to the treatment. The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain.

Test item-related histopathological alterations present in the kidneys of males treated at 1000 mg/kg bw/day consisted of an increased incidence and severity in hyaline droplet accumulation. The hyaline droplet accumulation recorded in the male kidneys was considered to relate to alpha-2u-globulin, a normal protein in male rats which undergoes reabsorption in the proximal cortical tubules. This male rat specific protein is not present in female rats nor in mammals, including man. Additionally, the minimal to slight degree severities, did not exceed the severities recorded for control males of the current study, and the hyaline droplet accumulation was not accompanied by indicators of tubular damage. This alteration was therefore regarded as non-adverse.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related gross pathological/macroscopic abnormalities detected. There were no microscopic neoplastic findings that were considered to be related to treatment with the test item.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

1. Thyroid Hormone Assessment:
Serum levels of T4 in F0-males were statistically significantly decreased in F0-males and F0 females at 300 and 1000 mg/kg bw/day (males: x0.8 and x0.8 of control, respectively and females: x0.8 and x0.7 of control, respectively). Mean T4 values remained within the historical control data range.

F0 generation :
Males Day 30 :
Control : T4 (ng/mL): mean (n=10) = 48.69 (SD: 6.26)
100 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 48.69 (SD: 7.86)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 40.47 (SD: 5.19)
1000 mg/kg bw/day: T4 (ng/mL): mean (n-10) = 40.04 (SD: 8.03)
Females Day 51 :
Control : T4 (ng/mL): mean (n=9) = 32.99 (SD: 9.61)
100 mg/kg bw/day: T4 (ng/mL): mean (n=8) = 28.38 (SD: 5.52)
300 mg/kg bw/day: T4 (ng/mL): mean (n=10) = 24.83 (SD: 4.66)
1000 mg/kg bw/day: T4 (ng/mL): mean (n=9) = 21.71 (SD: 2.49)

Historical control data for Wistar Han rats (period 2017 to 2020) - presented in the full study report :
F0-males: T4 (ng/mL): mean = 46.5 ; P5 – P95 = 31.30-65.20 (n=430).
F0 females: T4 (ng/mL): mean = 27.8 ; P5 – P95 = 16.70-40.40 (n=190).

Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item.

Additionally, F1 generation serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

Number of abortions:

no effects observed

Description (incidence and severity):

No signs of abortion or premature birth were observed within the study. No deficiencies in maternal care were noted.

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The number of implantations (implantation sites) was considered unaffected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study).

No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

It was considered offspring survival was not affected by treatment.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

Offspring survival was not affected by treatment. Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was 100% for all groups.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation length and gestation index were unaffected by treatment. Except for one female (#58; implantations only) at 100 mg/kg bw/day and one female (#78 humanely terminated in extremis due to difficulties during delivery) at 1000 mg/kg bw/day all pregnant females had live offspring. The gestation indices were respectively: 100%, 89%, 100% and 90% for the control, 100, 300 and 1000 mg/kg bw/day groups. The failed pregnancy/delivery of female #58 and #78, without related histopathology changes in reproductive organs were judged to be unrelated to treatment and of incidental occurrence, with no dose-response.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females showed normal estrous cycles before treatment. It was considered estrous cyclicity, pre-coital interval, fertility and mating performance were unaffected by treatment. Additionally, offspring survival was not affected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study).

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Abnormalities:

no effects observed

Description (incidence and severity):

No treatment related Adverse Effects observed up to the maximum dose level

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring body weight

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

There was no effect of parental treatment on offspring survival. Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was considered not to be affected by treatment with the test item. Viability indices were 100%, 96%, 100% and 100% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively. Three pups at 100 mg/kg bw/day from the same litter (female #60) were missing on PND 2. Missing offspring were most likely cannibalised. No toxicological relevance was attributed to these findings since the mortality incidence did not show a dose-related trend and within the expected range considered normal for pups of this age.

In addition, there were no toxicologically relevant macroscopic/pathological findings observed for offspring.

The number of implantations (implantation sites) was considered unaffected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study). No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring sex ratio

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on offspring body weight. The number of implantations (implantation sites) was considered unaffected by treatment. Live litter sizes were 10.3, 10.6, 11.3 and 8.9 living pups/litter for the respectively: control, 100, 300 and 1000 mg/kg bw/day groups. The lower mean number of living pups recorded at 1000 mg/kg bw/day was related to the higher post implantation survival index in this group. Specifically, the post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was respectively: 93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups. This lower post-implantation survival index in the high dose group could only partly be attributed to the failed delivery of female #78. Relationship to treatment could not be excluded (which was on a precautionary basis : reflected in the conclusion of the present screening study). No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant macroscopic/pathological findings observed for offspring.

Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. The statistically significantly higher mean for normalized anogenital distance of male pups at 300 mg/kg bw/day occurred without a dose response. This variation was considered not to be related to treatment. Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Visceral malformations:

no effects observed

Description (incidence and severity):

Macroscopic examination of offspring did not reveal any findings attributable to treatment.

Other effects:

no effects observed

Description (incidence and severity):

F1 offspring:
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item.

There were no other macroscopic findings finding observed in the offspring that were attributable to parental treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in litter size and weights

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index (relationship to treatment could not be excluded and therefore on a precautionary basis : reflected in the conclusion of the present screening study).

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 10-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by oral gavage administration for at least four weeks. Three treatment groups with a control were conducted, each comprising ten male and ten female rats which received oral gavage test item at doses of 0 (Control), 100, 300, 1000 mg/kg bw/day test item in corn oil vehicle.Chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity.Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 37-42 days. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received the vehicle, corn oil, at the same dose volume as treated groups.The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development: mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups). No treatment-related changes were noted in any of the following parameters investigated in this study, i.e. clinical appearance, functional observations, motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex, body weight, food consumption, clinical laboratory investigations (haematology, clotting parameters), and macroscopic examination. One female (no. 78) treated at 1000 mg/kg was humanely terminated (in extremis) on day 38 (during delivery). Due to difficulties during delivery (vaginal blood loss for 2 days with no pups delivered). Pregnancy for this female was confirmed based on palpation (no evidence of mating). Macroscopic observation revealed discolouration of the thyroid and a thymus reduced in size. Six implantation sites were noted, and the uterus contained six (6) live fetuses. Mortality was considered to be unrelated to treatment with the test item due to the incidental occurrence.

Test item-related morphologic alterations were present in the kidneys of males treated at 1000 mg/kg bw/day and consisted of an increased incidence and severity in hyaline droplet accumulation. The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha-2u-globulin. This male rat specific protein is not present in female rats nor in mammals, including man (ref: P.S. Sahota et al., 2013). The severities recorded at 1000 mg/kg bw/day (up to slight degree) did not exceed the severities recorded for control males of the current study, and the hyaline droplet accumulation was not accompanied by indicators of tubular damage and was therefore considered a non-adverse finding. Test item-related higher liver weights (absolute and relative to body weight) in males at 1000 mg/kg bw/day and increased alkaline phosphatase (ALP) in males (not achieving statistical significance) and females was recorded. Higher (absolute and relative to body weight) kidney weights were recorded in males at 300 and 1000 mg/kg bw/day. These effects observed in liver and kidney occurred without macroscopic and microscopic correlates and were therefore considered a non-adverse finding.

In males and females at 300 and 1000 mg/kg/day a test item related decrease in total T4 levels was recorded (in males 80% and 80% of control, respectively and in females 80% and 70% of control, respectively) . Although mean values remained within the historical control data range. Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item. Within reproductive parameters, no toxicologically significant changes were noted in this study, i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs.

In developmental parameters, a lower post implantation survival index was recorded at 1000 mg/kg bw/day (93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively). These changes could only partly be attributed to the female (no. 78) at 1000 mg/kg bw/day that was humanely sacrificed due to delivery difficulties. Even if the pups of this female were born, the post-implantation survival index would still be lower compared to the concurrent control. Therefore, a possible relation with treatment could not be excluded. As a consequence of the lower post-implantation survival index, litter size was also decreased in females treated at 1000 mg/kg bw/day. No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

 

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The key study is GLP compliant and of a high quality (Klimisch 1); The available information as a whole meets the tonnage driven information requirements of REACH.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive/developmental screening study – Oral:

Key study : OECD TG 422, 2021 :

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 10-day sighting study, the systemic toxic potential of the test item in rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints was conducted by oral gavage administration for at least four weeks. Three treatment groups with a control were conducted, each comprising ten male and ten female rats which received oral gavage test item at doses of 0 (Control), 100, 300, 1000 mg/kg bw/day test item in corn oil vehicle.Chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity.Males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 51-63 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 13 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 37-42 days. The offspring received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group was assigned to each phase, and received the vehicle, corn oil, at the same dose volume as treated groups.The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations, body weight and food consumption, estrous cycle determination, clinical pathology, measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations.

In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, pre-coital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development: mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups). No treatment-related changes were noted in any of the following parameters investigated in this study, i.e. clinical appearance, functional observations, motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex, body weight, food consumption, clinical laboratory investigations (haematology, clotting parameters), and macroscopic examination. One female (no. 78) treated at 1000 mg/kg was humanely terminated (in extremis) on day 38 (during delivery). Due to difficulties during delivery (vaginal blood loss for 2 days with no pups delivered). Pregnancy for this female was confirmed based on palpation (no evidence of mating). Macroscopic observation revealed discolouration of the thyroid and a thymus reduced in size. Six implantation sites were noted, and the uterus contained six (6) live fetuses. Mortality was considered to be unrelated to treatment with the test item due to the incidental occurrence.

Test item-related morphologic alterations were present in the kidneys of males treated at 1000 mg/kg bw/day and consisted of an increased incidence and severity in hyaline droplet accumulation. The hyaline droplet accumulation recorded in the male kidneys was considered to represent alpha-2u-globulin. This male rat specific protein is not present in female rats nor in mammals, including man (ref: P.S. Sahota et al., 2013). The severities recorded at 1000 mg/kg bw/day (up to slight degree) did not exceed the severities recorded for control males of the current study, and the hyaline droplet accumulation was not accompanied by indicators of tubular damage and was therefore considered a non-adverse finding. Test item-related higher liver weights (absolute and relative to body weight) in males at 1000 mg/kg bw/day and increased alkaline phosphatase (ALP) in males (not achieving statistical significance) and females was recorded. Higher (absolute and relative to body weight) kidney weights were recorded in males at 300 and 1000 mg/kg bw/day. These effects observed in liver and kidney occurred without macroscopic and microscopic correlates and were therefore considered a non-adverse finding.

In males and females at 300 and 1000 mg/kg/day a test item related decrease in total T4 levels was recorded (in males 80% and 80% of control, respectively and in females 80% and 70% of control, respectively) . Although mean values remained within the historical control data range. Thyroid Stimulating Hormone (TSH) values were considered unaffected by treatment with the test item. Within reproductive parameters, no toxicologically significant changes were noted in this study, i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs.

In developmental parameters, a lower post implantation survival index was recorded at 1000 mg/kg bw/day (93%, 91%, 92% and 78% for the control, 100, 300 and 1000 mg/kg bw/day groups, respectively). These changes could only partly be attributed to the female (no. 78) at 1000 mg/kg bw/day that was humanely sacrificed due to delivery difficulties. Even if the pups of this female were born, the post-implantation survival index would still be lower compared to the concurrent control. Therefore, a possible relation with treatment could not be excluded. As a consequence of the lower post-implantation survival index, litter size was also decreased in females treated at 1000 mg/kg bw/day. No treatment-related changes were noted in any other developmental parameters investigated, i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels, and macroscopic examination.

 

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) was regarded to be 1000 mg/kg bw/day bw/day for males/females. For reproduction the NOAEL was 1000 mg/kg bw/day and/or for developmental effects the NOAEL was 300 mg/kg bw/day based on apparent decreased post-implantation survival index.

Mode of Action Analysis / Human Relevance Framework

Not applicable.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for reproductive/developmental toxicity.

 

References:

1. ECHA Guidance on Application on the CLP Criteria, (v5.0, July 2017)