1-methoxy-4-[3-methyl-4-(2-phenylethoxy)but-3-en-1-yl]benzene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Oral (gavage): NOAEL (rat, systemic toxicity): ≥ 150 mg/kg bw/day, male/female, OECD TG 422, 2022
Oral: NOAEL (rat, reproductive toxicity): ≥ 150 mg/kg bw/day, male/female, based on female changes to: estrous cycle length and regularity, mean number of implantation, OECD TG 422, 2022
Oral: NOAEL (rat, developmental toxicity): ≥ 150 mg/kg bw/day based on apparent changes to: gestation index, decreased post-implantation survival index, male/female, OECD TG 422, 2022

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01-03-2021 to 21-02-2022

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 10, 30 and 100 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 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 5 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 5 hours at room temperature under normal light conditions and for at least 8 days in the refrigerator and/or at least 3 weeks in the freezer (at ≤ 15°C) 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 5 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. It was noted during the definitive study that test item sample analysis was based on version 2 calibration, with range 1 to 100 mg/mL. This recalibration (from analytical method used in the preliminary range finding study at 1 to 200 mg/mL) was prior to definitive test initiation due to analytical test system instability. Therefore, a second calibration was performed in range 1 to 100 mg/mL which yielded a coefficient of correlation (r) > 0.99 and mean recoveries of the QC samples were within the criterion range of 90-110%. All acceptability criteria were met (documented in the full study report).
- 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: 286 – 347 g; Females: 185 – 245 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 or 2000P type, height 21.5 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 or 2000P type, height 21.5 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: six days before commencement of treatment. Females: six days between arrival and start of estrous cycle smears (females) i.e. six 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 22)
- Humidity (%): 55 ± 15 (or 40 to 70 : actual 44 to 74%)
- Air changes (per hr): > 10 per hour (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2021-04-13 To: 2021-06-18

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 10, 30 and 100 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 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 5 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 10, 30 and 100 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], 50 mg/kg/bw [or 10 mg/mL], 150 mg/kg/bw [or 30 mg/mL], 500 mg/kg/bw [or 100 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 Day 44 of dosing the formulations were only acclimatized for 8 minutes after removal from the refrigerator. Deviation was of incidental nature, and was considered not to have adversely affected the study outcome. Separately, females #56 (Group 1), #67 and #69 (Group 2), #76 and #80 (Group 3), #90 and #92 (Group 4), were not dosed on one occasion as these females were littering at the designated dosing period. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

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 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 5 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. Version 2 of the analytical validation is provided here (see ‘-Other:’ below for further information). The reference item was prepared as a Quality Control (QC) solution at 1 mg/mL (Formulation A) and 250 mg/mL (Formulation B) in the vehicle according to internal procedure. Separately, six calibration solutions in the concentration range of 1 – 50.0 mg/L with acetonitrile from two analytical stock solutions at 2 to 100 mg/L and then water (dilution factor of 2). These in turn were prepared from two 1000 mg/L and 5000 mg/L standard stock and spiking solutions. The end solution of the calibration solutions was acetonitrile/water (50/50 v/v). 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) > 0.99. Accuracy and precision was confirmed in the range 85 – 115% with CoV < 10%. 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 ≤ 10%. 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 10, 30 and 100 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], 50 mg/kg/bw [or 10 mg/mL], 150 mg/kg/bw [or 30 mg/mL], 500 mg/kg/bw [or 100 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 acetonitrile 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: It was noted during the definitive study that test item sample analysis was based on version 2 calibration, with range 1 to 100 mg/mL. This recalibration (from analytical method used in the preliminary range finding study at 1 to 200 mg/mL) was prior to definitive test initiation due to analytical test system instability. Therefore, a second calibration was performed in range 1 to 100 mg/mL which yielded a coefficient of correlation (r) > 0.99 and mean recoveries of the QC samples were within the criterion range of 90-110%. All acceptability criteria were met (documented in the full study report).

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 14 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 44 days and/or total litter loss: 37 to 44 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.:

50 mg/kg bw/day (nominal)

Remarks:

Low – Group 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Intermediate – Group 3

Dose / conc.:

500 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) with satellite recovery groups (5 male / 5 female) at control and highest dose level only
F1 generation were not dosed (were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/faeces.

Males:
Control (toxicity test) = 10
Control (recovery phase) = 5
50 mg/kg bw/day (toxicity test) = 10
150 mg/kg bw/day (toxicity test) = 10
500 mg/kg bw/day (toxicity test) = 10
500 mg/kg bw/day (recovery phase) = 5

Females:
Control (toxicity test) = 10
Control (recovery phase) = 5
50 mg/kg bw/day (toxicity test) = 10
150 mg/kg bw/day (toxicity test) = 10
500 mg/kg bw/day (toxicity test) = 10
500 mg/kg bw/day (recovery phase) = 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this study (0, 50, 150 and 500 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). 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. (i) At 500 mg/kg bw/day: No mortality, with salivation in 1/3 females on Days 7 and 12-14, and in 1/3 females on Day 12 within clinical signs. Body weight losses in 2/3 females on Day 5 (2-7%) and in 1/3 females between Days 5-10 (2%). 2% body weight gain in 2/3 females on Day 14; the third female was back to start weight on Day 14. Food consumption was low over Days 1-5. Food consumption partly recovered over Days 5-10 and 10-14, but relative food consumption remained slightly low. No macroscopic abnormalities were noted at necropsy. High liver weights in 3/3 females. Kidney weights considered normal. (ii) At 1000 mg/kg bw/day: 1/3 females humanely sacrificed in extremis on Day 7. Hunched posture, piloerection, pale, dehydrated and lean appearance were recorded together with severe body weight loss (18%). Separately, within clinical signs: hunched posture in 2/2 females from Day 4 onwards. Piloerection in 2/2 females from Day 5 or 7 onwards. Salivation in 2/2 females on Days 7 and/or 13. Body weight losses in 2/2 females on Day 5 (7%). Body weight gain in 2/2 females on Day 10 (2-4%) and 14 (7-8%). Food consumption was extremely low over Days 1-5. Food consumption partly recovered over Days 5-10 and 10-14, but relative food consumption remained slightly low. During macroscopic examination at necropsy: stomach distended with gas, grey-white discolouration of jejunum and reduced size of thymus in 1/3 females (sacrificed in extremis on Day 7). No abnormalities noted in 2/3 females that survived up to scheduled necropsy. High liver and kidney weights were seen in 2/2 females. 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. Within the definitive test: the high-dose level should produce some toxic effects, but not death nor obvious suffering. The mid-dose level is expected to produce minimal to moderate toxic effects. The low-dose level should produce no observable indications of toxicity. Consequently, 50, 150 and 500 mg/kg bw/day by oral gavage were chosen. Applicant assessment indicates: at 1000 mg/kg bw/day there were humane sacrifices in extremis and/or significant clinical signs of toxicity together with significant body weight loss. At 500 mg/kg bw/day there were signs of toxicity: body weight loss, low food consumption. On this basis the highest dose was selected as 500 mg/kg bw/day within the OECD TG 422 test and/or is consistent with Regulation (EC) 1907/2006 as amended by Commission Regulation (EU) 2021/979 and/or that ‘the data generated are adequate for hazard identification and risk assessment’.
- Rationale for animal assignment (if not random): Randomly assigned.
- Fasting period before blood sampling for clinical biochemistry: F0-males (both Main and Recovery males) and Recovery females (except for animals which were sacrificed in extremis or found dead) : fasted overnight for a maximum of 24 hours before blood sampling. F0-Main (toxicity/repro) females will not be fasted overnight.
- Rationale for selecting satellite groups: Determine if toxic effects in males and females and F0 were recoverable.
- Post-exposure recovery period in satellite groups: 14 days
- 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: 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. Mated Main females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A terminal weight was recorded on the day of scheduled necropsy.

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 and Thyroid Stimulating Hormone (TSH) was conducted for F0-animals (both sexes) and measurement of total T4 was conducted for PND 14-16 pups. 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 any further selected males) 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: Yes. To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (if possible from one male and one female pup). Selective elimination of pups, e.g. based upon body weight or AGD, was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

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: 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)
- 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 14 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 44 days and/or total litter loss: 37 to 44 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: Brain ; Cervix #a ; Epididymis #b ; Gland, adrenal #b ; Gland, coagulation #b, #c ; Gland, parathyroid #d ; Gland, prostate ; Gland, seminal vesicle #b ;
Gland, thyroid #b ; Heart ; Kidney #b ; Liver ; Ovaries #b ; Spleen ; Testes #b ; Thymus ; Uterus
Where: #a : weighed together with the uterus ; #b : paired organ weight ; #c : weighed together with the seminal vesicles ; # d Weighed together with the thyroid.
For all Remaining Animals (including Females that Failed to Deliver Pups and Females with Total Litter Loss): Epididymis #a ; Gland, coagulation #a, #b ; Gland, parathyroid
#c ; Gland, prostate ; Gland, seminal vesicle #a ; Gland, thyroid #a ; Testes #a
Where: #a : paired organ weight ; #b : weighed together with the seminal vesicles ; # c Weighed together with the thyroid.
The numbers of former implantation sites were recorded for all paired Main females. In case no macroscopically visible implantation sites were present in Main females, nongravid 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.

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: Animal identification ; Artery, aorta ; Body cavity, nasopharynx ; Bone marrow ; Bone, femur ; Bone, sternum ; Brain (eight levels) ; Cervix ; Epididymides #a ; Esophagus ; Eye #a ; Gland, adrenal ; Gland, coagulation ; Gland, harderian #a ; Gland, lacrimal ; Gland, mammary ;
Gland, parathyroid #c ; Gland, pituitary ; Gland, prostate ; Gland, salivary ; Gland, seminal vesicle ; Gland, thyroid ; Gross lesions/masses ; Gut-associated lymphoid tissue ; Heart ; Kidney ; Large intestine, cecum ; Large intestine, colon ; Large intestine, rectum ; Larynx ; Liver ; Lung ; Lymph node (mandibular and mesenteric site) ; Muscle, skeletal ; Nerve, optic #b ; Nerve, sciatic ; Ovaries ; Pancreas ; Skin ; Small intestine, duodenum ; Small intestine, ileum ; Small intestine, jejunum ; Spinal cord ; Spleen ;
Stomach ; Testes #a ; Thymus ; Tongue ; Trachea ; Urinary bladder ; Uterus ; Vagina
Where: #a = Preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours ; #b = Only collected if present in the routine section of the eye. Part of the optic nerve attached to the eye was fixed in Modified Davidsons’s fixative. The remaining part of the optic nerve was placed in formalin ; #c = Only collected if present in the routine section of the thyroid.
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 : Animal identification ; Cervix ; Epididymis #a ; Gland, coagulation ;
Gland, mammary ; Gland, parathyroid #b ; Gland, pituitary ; Gland, prostate ; Gland, seminal vesicle ; Gland, thyroid ; Gross lesions/masses ; Ovaries ; Testes #a ; Uterus ;
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. In addition to blood collection, the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
- 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. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
- These animals were subjected to post-mortem 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.

- Other: Unscheduled mortality: Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

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:

effects observed, treatment-related

Description (incidence and severity):

At up to 500 mg/kg bw/day: Males: there were no significant clinical signs observed in Main and Recovery males related to treatment. Females: there were no significant clinical signs observed in Recovery females related to treatment. In Main females, one Main female (#93) showed piloerection, hunched posture, bleeding of the vaginal opening and red staining of the genital region after Day 10 post-coitum. These observations were possibly related to cysts in the ovaries, or possible signs of abortion and relationship to treatment could not be excluded.
At up to 150 mg/kg bw/day: Males and Females: there were no significant clinical signs observed.
All other signs (piloerection and/or hunched posture and/or salivation) in males and females were considered incidental findings unrelated to treatment. Salivation was considered to be a physiological response rather than a sign of systemic toxicity. All other signs were within the range of background findings for the species and strain of the test system.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

At up to 500 mg/kg bw/day: there were no treatment related mortalities. One Recovery (nulliparous) female (#97) was sacrificed in extremis. There were no test item-related microscopic findings. This was considered to be related to the blood sampling procedure. One Recovery (nulliparous) female (#100) was found dead in the cage on Day 9 of Recovery. No clinical signs or body weight changes related to mortality were observed in previous days. There were no test item-related microscopic findings. Necropsy findings (dark red fluid in the abdominal cavity, many reddish foci on the thymus and non-collapsed lungs) suggested this was related to the gavage procedure. The mortalities were considered non-treatment related.
At up to 150 mg/kg bw/day: Males and Females: there were no mortalities.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At up to 500 mg/kg bw/day: there were treatment-related changes in body weight although these were considered not adverse, considering the magnitude of the change (< 10% difference compared to control). Specifically:
(i) Males: In Main males, body weight gain was reduced when compared to the control group from Day 8 of treatment onwards at 500 mg/kg bw/day and 150 mg/kg bw/day (up to 83% and 36% lower than control, respectively). In Recovery males, body weight gain remained reduced up to the end of the Recovery Period (43% lower than control and not statistically significant on Day 14). No statistically significant effects on absolute body weight were noted, although a trend towards lower body weights was observed at 150 and 500 mg/kg bw/day (3 and 4%, respectively) on day 15 of the reproduction period. Body weights of Recovery males were 5% lower than control on Day 14 of the recovery period.
(ii) Females: In Main females, body weight gain was reduced from post-coitum Day 11 onwards. On Day 17 and 20, this was also reflected in a significant decrease in absolute body weight (up to 16% lower than control). During lactation no differences in body weight gain were recorded. A trend towards lower absolute body weight in females at 500 mg/kg bw/day, when compared to control females remained present during the lactation period (up to 5% lower than control). In Recovery (nulliparous) females at 500 mg/kg bw/day no toxicologically relevant changes in body weight or body weight gain were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At up to 500 mg/kg bw/day: Males: there were no statistically significant test item related effects on food consumption. Females: absolute (but not relative) food consumption was reduced from post-coitum Day 11 onwards (up to 14% lower than control). During lactation, absolute and relative food consumption in Main females were reduced (up to 30% lower than control for absolute food consumption, statistically significant on Days 4-7 and 7-13 only). In Recovery (nulliparous) females no toxicologically relevant differences were noted.
At up to 150 mg/kg bw/day: there were no statistically significant test item related effects on food consumption of males or females.

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 of the control (#59) at the end of treatment and one female (#80) at 150 mg/kg bw/day only post termination. Exophthalmus, was also seen in one female (#97) at 500 mg/kg bw/day that was terminated early. (#97 was terminated in extremis see ‘- Mortality’ field above, mortality was considered related to the blood sampling procedure).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At up to 500 mg/kg bw/day: Males: Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects. Females: Main (lactating) females: Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects. Specifically: Lower eosinophil concentrations were considered the result of a slightly high control value (HCD range: documented in the full study report). In Recovery (nulliparous) females: increased white blood cell count (WBC, x1.59 of control) corresponding with increased lymphocyte counts (x1.53 of control) which recovered after the 14-day treatment-free period. Higher red blood cell distribution width (RDWG, x1.07 of control) which remained present after the 14-day treatment-free period (x1.11 of control). Lower mean corpuscular volume (MCV, 0.96x of control) which remained present after the 14-day treatment-free period (x0.96 of control). Lower mean corpuscular hemoglobin (MCH) concentrations in Recovery (nulliparous) females at 500 mg/kg bw/day present after the 14-day treatment-free period was considered unrelated to treatment as they occurred after the treatment free period only. It was considered, in the absence of corroborating findings the haematological changes were considered non-adverse. There were no effects on coagulation parameters within the study.
At up to 150 mg/kg bw/day: Males and Females: Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects.

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).

At up to 500 mg/kg bw/day: Males: Increased alanine aminotransferase (ALT) activity (up to x1.34 of control) which was recovered after the 14-day treatment-free period (x1.18 of control). Increased total bilirubin concentration (x1.55 of control) [with a similar trend at 150 mg/kg bw/day (1.20x of control, individual values generally within the same range as control and not statistically significant)], and a decreased total bilirubin at the end of the recovery period (0.80x of control). Females: Increased total bilirubin concentration in Recovery (nulliparous) females (x2.20 of control) which was recovered after the 14-day treatment-free period (x0.85 of control). For males/females: all parameters recovered during the Recovery Period. There were no corroborating findings and therefore these changes were considered non-adverse. All other findings were considered unrelated to treatment.
At up to 150 mg/kg bw/day: Males and Females: Assessment of clinical chemistry parameters at the end of the treatment period revealed no treatment related effects.

Endocrine findings:

no effects observed

Description (incidence and severity):

1. Thyroid Hormone Assessment:
(i) F0 generation :
Serum levels of T4 in F0-males and F0-females at the end of the treatment period revealed no treatment related statistically significant effects.
Specifically, at 500 mg/kg bw/day in males: a trend towards lower mean serum T4 levels was recorded, although the magnitude of change was < 20% relative to the controls and therefore this was not considered test item related. Furthermore, applicant assessment also indicates, only one individual at 500 mg/kg bw/day (male #37) had a T4 serum level outside the control range.
(ii) F1 offspring: Serum T4 levels in male and female PND 14 pups were not considered to be affected by treatment with the test item up to 150 mg/kg bw/day. (Since only 5 females in the high dose at 500 mg/kg bw/day group ultimately delivered pups: full toxicological evaluation could not be performed except for gestation and post-implantation survival index).

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):

At up to 500 mg/kg bw/day: Males: for the liver: centrilobular hepatocellular hypertrophy (minimal) 1/10 and/or (slight) in 9/10 Main males which correlated with macroscopically enlarged liver and/or higher liver weights. There were no test item-related microscopic findings in the liver after the 14-day recovery period.
Females: for the liver: centrilobular hepatocellular hypertrophy (minimal) 1/10 and/or (slight) in 2/10 Main females which correlated with higher liver weights. There were no test item-related microscopic findings in the liver after the 14-day recovery period.
At up to 150 mg/kg bw/day: Males: for the liver: centrilobular hepatocellular hypertrophy (minimal level) was recording in 4/5 Main males which correlated with higher liver weight. There were no test item-related microscopic findings in the liver after the 14-day recovery period. Females: No test item-related microscopic findings.
The liver effects/changes were interpreted to be of initial concern due to the high magnitude of liver weight increase but were considered non-adverse. This was given the minimal to slight degree of microscopic hypertrophy, absence of any accompanying inflammatory, degenerative or necrotic changes, and reversibility of the microscopic findings and/or organ weight after the treatment-free recovery period (See ‘Organ weight findings’ for further information).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

There were no relevant microscopic neoplastic findings that were considered to be related to treatment with the test item.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At up to 500 mg/kg bw/day:
(i) Estrous: Main females: four did not have a regular estrous cycle (#90, #92, #93 and #94). Female #90 was not pregnant, and female #93 and #94 had implantation sites only. Female #92 did have a litter. Relationships to treatment with the test item could not be excluded and at the incidence observed was regarded adverse. Recovery (nulliparous) females (at 500 mg/kg bw/day) estrous cycle length and regularity were unaffected by treatment with the test item.
(ii) Gestation index (females with living pups on Day 1 compared to the number of pregnant females) : was markedly lower. Only 5/8 pregnant females delivered; whereas the other three females had implantation sites only. This was considered treatment-related. The gestation indices were for control, 50, 150 and 500 mg/kg bw/day: 100%, 100%. 100% and 63%, respectively.
(iii) Gestation length: the gestation periods were considered unaffected by treatment. For the five females with litter, a gestation period of 22.2 days was recorded compared to 21.4 days in the control group, which was considered within normal ranges of biological variation.
(iv) Number of implantation sites: mean number of implantation sites was lower compared to the control group. Two females (#89 and #94) had a single implantation site only. This was considered related to treatment with the test item and considering the magnitude of change this was regarded adverse. Mean number of implantation sites for the control, 50, 150 and 500 mg/kg bw/day groups were: 13.6, 10.5, 13.3 and 5.8, respectively.
At up to 150 mg/kg bw/day:
(i) Estrous: Main females: estrous cycle: length and regularity were unaffected by treatment. Gestation index (females with living pups on Day 1 compared to the number of pregnant females) was considered not to be affected by treatment with the test item.
(ii) Gestation index (females with living pups on Day 1 compared to the number of pregnant females): was considered not to be affected by treatment with the test item.
(iii) Gestation length: was considered not to be affected by treatment with the test item.
(iv) Number of implantation sites: was considered not to be affected by treatment with the test item.

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. The 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. No cell or stage specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

At up to 500 mg/kg bw/day: there was no effect on fertility or mating performance related to treatment.
(i) Mating index: for the control, 50, 150 and 500 mg/kg bw/day groups the mating indices were: 100%, 100%, 90% and 100%, respectively. Mating index was considered not to be affected by treatment with the test item.
(ii) Fertility index: for the control, 50, 150 and 500 mg/kg bw/day groups the fertility indices were: 100%, 100%, 89% and 80%, respectively. At the high dose group level, the fertility index was slightly low (80%). A total of two females were not pregnant. As this was considered to be within the normal ranges of biological variation, it was regarded not toxicologically relevant. Fertility index was considered not to be affected by treatment with the test item.

The applicant derives the relevant NOAELs from the information and/or conclusions given within the study report, as follows:

Oral (gavage): NOAEL (rat, systemic toxicity): ≥ 150 mg/kg bw/day, male/female, OECD TG 422, 2022
Oral: NOAEL (rat, reproductive toxicity): ≥ 150 mg/kg bw/day, male/female, based on female changes to: estrous cycle length and regularity, mean number of implantation, OECD TG 422, 2022
Oral: NOAEL (rat, developmental toxicity): ≥ 150 mg/kg bw/day based on apparent changes to: gestation index, decreased post-implantation survival index, male/female, OECD TG 422, 2022

There were numerous systemic toxicity observations within the study at the high dose (500 mg/kg bw/day), specifically:

1. Lower body weight in males
2. Lower body weight gain in males
3. Lower body weight in females
4. Increased serum alanine aminotransferase in males
5. Increased bilirubin in males
6. Increased sodium, potassium, and calcium in males
7. Enlarged liver (possibly adaptive rather than adverse)
8. Increased kidney weight in males (absolute and relative)
9. Increased uterus weight in females (absolute and relative)
10. Increased incidence of Grade 2 hepatocyte hypertrophy in males and females.

Individually, none of the listed effects stand out as remarkable. However, collectively they could be considered as adverse and demonstrate that exposure to the high dose (500 mg/kg bw/day) of the test item is not well tolerated and/or such effects were not observed in the low (50 mg/kg bw/day) and middle (150 mg/kg bw/day) dose groups.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

organ weights and organ / body weight ratios

reproductive function (oestrous cycle)

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

Note: at 500 mg/kg bw/day, full (developmental) toxicological evaluation could not be performed (except for gestation and post-implantation survival index) since data was available of only 5 females in the high dose group.
At up to 150 mg/kg bw/day: Clinical signs: was considered not to be affected by parental treatment with the test item.

Mortality / viability:

no mortality observed

Description (incidence and severity):

At up to 150 mg/kg bw/day: There were no treatment related mortality associated with treatment within F1 males or females.
(i) Live birth index (number of live offspring on Day 1 after littering compared to the total number of offspring born): was considered not to be affected by treatment with the test item. Live birth indices for the control, 50 and 150 mg/kg bw/day groups were: 100%. 99% and 99%. One pup at 50 mg/kg bw/day and one pup at 150 mg/kg bw/day were found dead during first litter check. For the pup at 50 mg/kg/day no milk was recorded in the stomach. For the pup at 150 mg/kg/day cannibalism of the abdominal region was recorded.
(ii) Viability index (number of live offspring on PND4): was considered not to be affected by treatment with the test item. Viability indices for the control, 50 and 150 mg/kg bw/day groups were: 100%. 100% and 99%. At 150 mg/kg bw/day one pup was missing on PND 4. No toxicological relevance was attributed to this missing pup since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
(iii) Macroscopic findings: it was considered there were no toxicologically relevant macroscopic/pathological findings observed for offspring. One male F1 pup of Main female #68 (at 50 mg/kg bw/day) had a small left epididymis and testis. At the incidence observed, this was considered not related to treatment with the test item.
(iv) Bodyweight: was considered not to be affected by parental treatment with the test item.
(v) Lactation index: the number of live offspring on PND13 compared to the number of offspring on PND4 was considered not to be affected by parental treatment with the test item. The lactation indices were for control, 50 and 150 mg/kg bw/day: 100%, 100% and 100%, respectively.
(vi) Clinical signs: was considered not to be affected by parental treatment with the test item.
(vii) No other toxicologically significant changes were noted in any of the remaining developmental parameters investigated in this study (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At up to 150 mg/kg bw/day: Bodyweight: was considered not to be affected by parental treatment with the test item.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

At up to 150 mg/kg bw/day: There was no effect of parental treatment on offspring anogenital distance.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

At up to 150 mg/kg bw/day: There was no effect of parental treatment no effect on areola/nipple retention. For the examined male pups, no nipples were observed at PND 13.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At up to 150 mg/kg bw/day: It was considered there were no toxicologically relevant macroscopic/pathological findings observed for offspring. One male F1 pup of Main female #68 (at 50 mg/kg bw/day) had a small left epididymis and testis. At the incidence observed, this was considered not related to treatment with the test item.

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:
(i) F0 generation :
Serum levels of T4 in F0-males and F0-females at the end of the treatment period revealed no treatment related statistically significant effects.
Specifically, at 500 mg/kg bw/day in males: a trend towards lower mean serum T4 levels was recorded, although the magnitude of change was < 20% relative to the controls and therefore this was not considered test item related. Furthermore, applicant assessment also indicates, only one individual at 500 mg/kg bw/day (male #37) had a T4 serum level outside the control range.
(ii) F1 offspring: Serum T4 levels in male and female PND 14 pups were not considered to be affected by treatment with the test item up to 150 mg/kg bw/day. (Since only 5 females in the high dose at 500 mg/kg bw/day group ultimately delivered pups: full toxicological evaluation could not be performed except for gestation and post-implantation survival index).

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is considered to be ≥ 150 mg/kg bw/day for males/females. For reproduction the NOAEL was ≥ 150 mg/kg bw/day based on female changes to: estrous cycle length and regularity, mean number of implantations and/or for developmental effects the NOAEL was ≥ 150 mg/kg bw/day based on apparent changes to: gestation index, decreased post-implantation survival index.

Executive summary:

The study was performed according to 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), 50, 150, 500 mg/kg bw/day test item in corn oil vehicle. Satellite recovery groups at 0 (control) and/or 500 mg/kg bw/day comprising five male and five female rats were also included to study the potential reversibility of possible toxic effects after a 14-day treatment-free period. Recovery animals were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity. The 10-day sighting study (preliminary toxicity test) was conducted using Group 1: 500 mg/kg bw/day (in corn oil), Group 2: 1000 mg/kg bw/day (in corn oil). Therein, at 1000 mg/kg bw/day there were humane sacrifices in extremis and/or significant clinical signs of toxicity together with significant body weight loss. At 500 mg/kg bw/day there were signs of toxicity: body weight loss, low food consumption, but not death nor obvious suffering. The definitive test dose range was therefore selected in accordance with the relevant guideline and legislative requirements. Within the definitive study, chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity. Males were treated for a minimum of 28 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 at least 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. Main Females were not dosed during littering. Recovery males/females were treated during the same period as Main males/females until at least the first scheduled necropsy of Main male/females. The offspring received no direct administration of the test item; any exposure was in utero or via the milk and/or from exposure to maternal urine/feces. 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). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Systemic toxicity parameters
There were no treatment related mortalities. At 150 and 500 mg/kg bw/day in males, treatment-related changes in body weight gain were observed from Day 8 of treatment onwards, which only partly recovered during the recovery period. Considering the absence of a significant effect on absolute body weights (<10% difference compared to control) this was considered not adverse. In Main females, at 500 mg/kg bw/day body weight gain was reduced from post-coitum Day 11 onwards, resulting in a statistically significant decrease in absolute body weight. This was possibly related to the lower litter size in this group. A trend towards lower absolute body weight in females at 500 mg/kg bw/day compared to control females remained present during the lactation period, but regarding the magnitude of change (<10% difference compared to control) regarded not adverse. At 500 mg/kg bw/day, test item-related lower food consumption in Main females during the post-coitum and lactation phase was possibly related to the lower litter size in this group. At 500 mg/kg bw/day in Recovery females: hematology parameters were affected by treatment which included: increased white blood cell and lymphocyte counts (recovered after the 14-day treatment-free period) and increased red blood cell distribution width (remained present after the recovery period); and decreased mean corpuscular volume (remained present after the recovery period). In the absence of corroborating findings these changes were considered non-adverse. At 500 mg/kg bw/day: clinical biochemistry parameters were affected by treatment including in males: increased total bilirubin, potassium, and calcium concentrations, and increased total bilirubin in females, at the end of treatment. All parameters recovered during the Recovery Period. There were no corroborating findings and therefore these changes were considered non-adverse. No treatment related significant changes in T4 concentrations were observed in any parental treatment group. Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations for the liver. There were test item-related higher liver weights in Main males starting at 150 mg/kg bw/day and in Main females at 500 mg/kg bw/day. For males at 500 mg/kg bw/day the higher liver weight correlated with the macroscopically enlarged liver and/or the higher liver weight correlated with microscopic centrilobular hepatocellular hypertrophy in Main males: 150 mg/kg bw/day (minimal degree) and Main males and Main females at 500 mg/kg bw/day (minimal to slight degree). At 500 mg/kg bw/day in Main males, the minor but statistically significant increase in ALT (alanine aminotransferase) noted at the end of the treatment period may correlate with higher liver weight and hepatocellular hypertrophy. Although was considered non-adverse. For males, there was complete recovery after the 14-Day treatment-free period for the liver findings, but for females there still was a significantly higher liver weight (relative to body weight only). For males, the changes were interpreted to be non-adverse given the minimal to slight degree of microscopic hypertrophy, absence of any accompanying inflammatory, degenerative or necrotic changes, and reversibility of the microscopic findings and/or organ weight after the 14-Day treatment-free recovery period. For females, non-adverse given the absence of correlating macroscopic or microscopic findings in the liver, the low magnitude of organ weight change and because the higher liver weight was only significant relative to body weight. In addition, there were no clear correlating clinical pathology changes. At 500 mg/kg bw/day, there was significant higher kidney weights in Main males and in Recovery females although no gross or microscopic correlations and therefore it was considered non-adverse. Higher uterus weight in Main females was without a clear microscopic correlates and recovered after the 14-Day treatment-free period and was therefore considered non-adverse. There was a significant higher spleen weight in Main females treated at 150 mg/kg bw/day and in Recovery females treated at 500 mg/kg bw/day. There were no gross observations in Main or Recovery females and no changes in hematology values. This was considered non-adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters and male T4 thyroid hormone levels).

Reproductive toxicity parameters
At 500 mg/kg bw/day, estrous cycle length and regularity were affected in 4/10 Main females. 4/10 females did not have a regular cycle. One of these four females was not pregnant, and two females had implantation sites only. Mean number of implantations sites was considered low and treatment-related given the magnitude of change this was regarded adverse. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, fertility index, spermatogenic profiling, and histopathological examination of reproductive organs). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Developmental toxicity parameters
At 500 mg/kg bw/day, full toxicological evaluation could not be performed (except for gestation and post-implantation survival index) since data was available of only 5 females in the high dose group. At 500 mg/kg bw/day, a test item-related decrease in gestation index was observed. 3/8 pregnant females had implantation sites only. One of the females showed bleeding of the vaginal opening and red staining of the genital region, which were considered potentially related to cysts in the ovaries, or possible signs of abortion. The lower gestation index was considered adverse. Furthermore, dose-related lower post-implantation survival index was recorded. Considering the magnitude of change, this was regarded adverse. At up to 150 mg/kg bw/day, no other toxicologically significant changes were noted in any of the remaining developmental parameters (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The applicant derives the relevant NOAELs from the information and/or conclusions given within the study report, as follows:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be ≥ 150 mg/kg bw/day for males/females. For reproduction the NOAEL was ≥ 150 mg/kg bw/day and/or for developmental effects the NOAEL was ≥ 150 mg/kg bw/day.

There were numerous systemic toxicity observations within the study at the high dose (500 mg/kg bw/day), specifically:

1. Lower body weight in males
2. Lower body weight gain in males
3. Lower body weight in females
4. Increased serum alanine aminotransferase in males
5. Increased bilirubin in males
6. Increased sodium, potassium, and calcium in males
7. Enlarged liver (possibly adaptive rather than adverse)
8. Increased kidney weight in males (absolute and relative)
9. Increased uterus weight in females (absolute and relative)
10. Increased incidence of Grade 2 hepatocyte hypertrophy in males and females.

Individually, none of the listed effects stand out as remarkable. However, collectively they could be considered as adverse and demonstrate that exposure to the high dose (500 mg/kg bw/day) of the test item is not well tolerated and/or such effects were not observed in the low (50 mg/kg bw/day) and middle (150 mg/kg bw/day) dose groups.

 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 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, 2022 : The study was performed according to 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), 50, 150, 500 mg/kg bw/day test item in corn oil vehicle. Satellite recovery groups at 0 (control) and/or 500 mg/kg bw/day comprising five male and five female rats were also included to study the potential reversibility of possible toxic effects after a 14-day treatment-free period. Recovery animals were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity. The 10-day sighting study (preliminary toxicity test) was conducted using Group 1: 500 mg/kg bw/day (in corn oil), Group 2: 1000 mg/kg bw/day (in corn oil). Therein, at 1000 mg/kg bw/day there were humane sacrifices in extremis and/or significant clinical signs of toxicity together with significant body weight loss. At 500 mg/kg bw/day there were signs of toxicity: body weight loss, low food consumption, but not death nor obvious suffering. The definitive test dose range was therefore selected in accordance with the relevant guideline and legislative requirements. Within the definitive study, chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity. Males were treated for a minimum of 28 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 at least 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. Main Females were not dosed during littering. Recovery males/females were treated during the same period as Main males/females until at least the first scheduled necropsy of Main male/females. The offspring received no direct administration of the test item; any exposure was in utero or via the milk and/or from exposure to maternal urine/feces. 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). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Systemic toxicity parameters
There were no treatment related mortalities. At 150 and 500 mg/kg bw/day in males, treatment-related changes in body weight gain were observed from Day 8 of treatment onwards, which only partly recovered during the recovery period. Considering the absence of a significant effect on absolute body weights (<10% difference compared to control) this was considered not adverse. In Main females, at 500 mg/kg bw/day body weight gain was reduced from post-coitum Day 11 onwards, resulting in a statistically significant decrease in absolute body weight. This was possibly related to the lower litter size in this group. A trend towards lower absolute body weight in females at 500 mg/kg bw/day compared to control females remained present during the lactation period, but regarding the magnitude of change (<10% difference compared to control) regarded not adverse. At 500 mg/kg bw/day, test item-related lower food consumption in Main females during the post-coitum and lactation phase was possibly related to the lower litter size in this group. At 500 mg/kg bw/day in Recovery females: hematology parameters were affected by treatment which included: increased white blood cell and lymphocyte counts (recovered after the 14-day treatment-free period) and increased red blood cell distribution width (remained present after the recovery period); and decreased mean corpuscular volume (remained present after the recovery period). In the absence of corroborating findings these changes were considered non-adverse. At 500 mg/kg bw/day: clinical biochemistry parameters were affected by treatment including in males: increased total bilirubin, potassium, and calcium concentrations, and increased total bilirubin in females, at the end of treatment. All parameters recovered during the Recovery Period. There were no corroborating findings and therefore these changes were considered non-adverse. No treatment related significant changes in T4 concentrations were observed in any parental treatment group. Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations for the liver. There were test item-related higher liver weights in Main males starting at 150 mg/kg bw/day and in Main females at 500 mg/kg bw/day. For males at 500 mg/kg bw/day the higher liver weight correlated with the macroscopically enlarged liver and/or the higher liver weight correlated with microscopic centrilobular hepatocellular hypertrophy in Main males: 150 mg/kg bw/day (minimal degree) and Main males and Main females at 500 mg/kg bw/day (minimal to slight degree). At 500 mg/kg bw/day in Main males, the minor but statistically significant increase in ALT (alanine aminotransferase) noted at the end of the treatment period may correlate with higher liver weight and hepatocellular hypertrophy. Although was considered non-adverse. For males, there was complete recovery after the 14-Day treatment-free period for the liver findings, but for females there still was a significantly higher liver weight (relative to body weight only). For males, the changes were interpreted to be non-adverse given the minimal to slight degree of microscopic hypertrophy, absence of any accompanying inflammatory, degenerative or necrotic changes, and reversibility of the microscopic findings and/or organ weight after the 14-Day treatment-free recovery period. For females, non-adverse given the absence of correlating macroscopic or microscopic findings in the liver, the low magnitude of organ weight change and because the higher liver weight was only significant relative to body weight. In addition, there were no clear correlating clinical pathology changes. At 500 mg/kg bw/day, there was significant higher kidney weights in Main males and in Recovery females although no gross or microscopic correlations and therefore it was considered non-adverse. Higher uterus weight in Main females was without a clear microscopic correlates and recovered after the 14-Day treatment-free period and was therefore considered non-adverse. There was a significant higher spleen weight in Main females treated at 150 mg/kg bw/day and in Recovery females treated at 500 mg/kg bw/day. There were no gross observations in Main or Recovery females and no changes in hematology values. This was considered non-adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters and male T4 thyroid hormone levels).

Reproductive toxicity parameters
At 500 mg/kg bw/day, estrous cycle length and regularity were affected in 4/10 Main females. 4/10 females did not have a regular cycle. One of these four females was not pregnant, and two females had implantation sites only. Mean number of implantations sites was considered low and treatment-related given the magnitude of change this was regarded adverse. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, fertility index, spermatogenic profiling, and histopathological examination of reproductive organs). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Developmental toxicity parameters
At 500 mg/kg bw/day, full toxicological evaluation could not be performed (except for gestation and post-implantation survival index) since data was available of only 5 females in the high dose group. At 500 mg/kg bw/day, a test item-related decrease in gestation index was observed. 3/8 pregnant females had implantation sites only. One of the females showed bleeding of the vaginal opening and red staining of the genital region, which were considered potentially related to cysts in the ovaries, or possible signs of abortion. The lower gestation index was considered adverse. Furthermore, dose-related lower post-implantation survival index was recorded. Considering the magnitude of change, this was regarded adverse. At up to 150 mg/kg bw/day, no other toxicologically significant changes were noted in any of the remaining developmental parameters (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The applicant derives the relevant NOAELs from the information and/or conclusions given within the study report, as follows:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be ≥ 150 mg/kg bw/day for males/females. For reproduction the NOAEL was ≥ 150 mg/kg bw/day and/or for developmental effects the NOAEL was ≥ 150 mg/kg bw/day.

There were numerous systemic toxicity observations within the study at the high dose (500 mg/kg bw/day), specifically:

1. Lower body weight in males
2. Lower body weight gain in males
3. Lower body weight in females
4. Increased serum alanine aminotransferase in males
5. Increased bilirubin in males
6. Increased sodium, potassium, and calcium in males
7. Enlarged liver (possibly adaptive rather than adverse)
8. Increased kidney weight in males (absolute and relative)
9. Increased uterus weight in females (absolute and relative)
10. Increased incidence of Grade 2 hepatocyte hypertrophy in males and females.

Individually, none of the listed effects stand out as remarkable. However, collectively they could be considered as adverse and demonstrate that exposure to the high dose (500 mg/kg bw/day) of the test item is not well tolerated and/or such effects were not observed in the low (50 mg/kg bw/day) and middle (150 mg/kg bw/day) dose groups

Effects on developmental toxicity

Description of key information

Oral (gavage): NOAEL (rat, systemic toxicity): ≥ 150 mg/kg bw/day, male/female, OECD TG 422, 2022
Oral: NOAEL (rat, reproductive toxicity): ≥ 150 mg/kg bw/day, male/female, based on female changes to: estrous cycle length and regularity, mean number of implantation, OECD TG 422, 2022
Oral: NOAEL (rat, developmental toxicity): ≥ 150 mg/kg bw/day based on apparent changes to: gestation index, decreased post-implantation survival index, male/female, OECD TG 422, 2022

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 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, 2022 : The study was performed according to 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), 50, 150, 500 mg/kg bw/day test item in corn oil vehicle. Satellite recovery groups at 0 (control) and/or 500 mg/kg bw/day comprising five male and five female rats were also included to study the potential reversibility of possible toxic effects after a 14-day treatment-free period. Recovery animals were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity. The 10-day sighting study (preliminary toxicity test) was conducted using Group 1: 500 mg/kg bw/day (in corn oil), Group 2: 1000 mg/kg bw/day (in corn oil). Therein, at 1000 mg/kg bw/day there were humane sacrifices in extremis and/or significant clinical signs of toxicity together with significant body weight loss. At 500 mg/kg bw/day there were signs of toxicity: body weight loss, low food consumption, but not death nor obvious suffering. The definitive test dose range was therefore selected in accordance with the relevant guideline and legislative requirements. Within the definitive study, chemical analyses of formulations were conducted once during the study to assess accuracy, and homogeneity. Males were treated for a minimum of 28 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 at least 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. Main Females were not dosed during littering. Recovery males/females were treated during the same period as Main males/females until at least the first scheduled necropsy of Main male/females. The offspring received no direct administration of the test item; any exposure was in utero or via the milk and/or from exposure to maternal urine/feces. 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). Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

Systemic toxicity parameters
There were no treatment related mortalities. At 150 and 500 mg/kg bw/day in males, treatment-related changes in body weight gain were observed from Day 8 of treatment onwards, which only partly recovered during the recovery period. Considering the absence of a significant effect on absolute body weights (<10% difference compared to control) this was considered not adverse. In Main females, at 500 mg/kg bw/day body weight gain was reduced from post-coitum Day 11 onwards, resulting in a statistically significant decrease in absolute body weight. This was possibly related to the lower litter size in this group. A trend towards lower absolute body weight in females at 500 mg/kg bw/day compared to control females remained present during the lactation period, but regarding the magnitude of change (<10% difference compared to control) regarded not adverse. At 500 mg/kg bw/day, test item-related lower food consumption in Main females during the post-coitum and lactation phase was possibly related to the lower litter size in this group. At 500 mg/kg bw/day in Recovery females: hematology parameters were affected by treatment which included: increased white blood cell and lymphocyte counts (recovered after the 14-day treatment-free period) and increased red blood cell distribution width (remained present after the recovery period); and decreased mean corpuscular volume (remained present after the recovery period). In the absence of corroborating findings these changes were considered non-adverse. At 500 mg/kg bw/day: clinical biochemistry parameters were affected by treatment including in males: increased total bilirubin, potassium, and calcium concentrations, and increased total bilirubin in females, at the end of treatment. All parameters recovered during the Recovery Period. There were no corroborating findings and therefore these changes were considered non-adverse. No treatment related significant changes in T4 concentrations were observed in any parental treatment group. Relationships were suspected between gross necropsy, organ weight, clinical pathology, and histopathology observations for the liver. There were test item-related higher liver weights in Main males starting at 150 mg/kg bw/day and in Main females at 500 mg/kg bw/day. For males at 500 mg/kg bw/day the higher liver weight correlated with the macroscopically enlarged liver and/or the higher liver weight correlated with microscopic centrilobular hepatocellular hypertrophy in Main males: 150 mg/kg bw/day (minimal degree) and Main males and Main females at 500 mg/kg bw/day (minimal to slight degree). At 500 mg/kg bw/day in Main males, the minor but statistically significant increase in ALT (alanine aminotransferase) noted at the end of the treatment period may correlate with higher liver weight and hepatocellular hypertrophy. Although was considered non-adverse. For males, there was complete recovery after the 14-Day treatment-free period for the liver findings, but for females there still was a significantly higher liver weight (relative to body weight only). For males, the changes were interpreted to be non-adverse given the minimal to slight degree of microscopic hypertrophy, absence of any accompanying inflammatory, degenerative or necrotic changes, and reversibility of the microscopic findings and/or organ weight after the 14-Day treatment-free recovery period. For females, non-adverse given the absence of correlating macroscopic or microscopic findings in the liver, the low magnitude of organ weight change and because the higher liver weight was only significant relative to body weight. In addition, there were no clear correlating clinical pathology changes. At 500 mg/kg bw/day, there was significant higher kidney weights in Main males and in Recovery females although no gross or microscopic correlations and therefore it was considered non-adverse. Higher uterus weight in Main females was without a clear microscopic correlates and recovered after the 14-Day treatment-free period and was therefore considered non-adverse. There was a significant higher spleen weight in Main females treated at 150 mg/kg bw/day and in Recovery females treated at 500 mg/kg bw/day. There were no gross observations in Main or Recovery females and no changes in hematology values. This was considered non-adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), clotting parameters and male T4 thyroid hormone levels).

Reproductive toxicity parameters
At 500 mg/kg bw/day, estrous cycle length and regularity were affected in 4/10 Main females. 4/10 females did not have a regular cycle. One of these four females was not pregnant, and two females had implantation sites only. Mean number of implantations sites was considered low and treatment-related given the magnitude of change this was regarded adverse. No treatment-related changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating index, precoital time, fertility index, spermatogenic profiling, and histopathological examination of reproductive organs). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Developmental toxicity parameters
At 500 mg/kg bw/day, full toxicological evaluation could not be performed (except for gestation and post-implantation survival index) since data was available of only 5 females in the high dose group. At 500 mg/kg bw/day, a test item-related decrease in gestation index was observed. 3/8 pregnant females had implantation sites only. One of the females showed bleeding of the vaginal opening and red staining of the genital region, which were considered potentially related to cysts in the ovaries, or possible signs of abortion. The lower gestation index was considered adverse. Furthermore, dose-related lower post-implantation survival index was recorded. Considering the magnitude of change, this was regarded adverse. At up to 150 mg/kg bw/day, no other toxicologically significant changes were noted in any of the remaining developmental parameters (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

The applicant derives the relevant NOAELs from the information and/or conclusions given within the study report, as follows:

Under the conditions of this study, the No-Observed-Adverse-Effect-Level (NOAEL) is considered to be ≥ 150 mg/kg bw/day for males/females. For reproduction the NOAEL was ≥ 150 mg/kg bw/day and/or for developmental effects the NOAEL was ≥ 150 mg/kg bw/day.

There were numerous systemic toxicity observations within the study at the high dose (500 mg/kg bw/day), specifically:

1. Lower body weight in males
2. Lower body weight gain in males
3. Lower body weight in females
4. Increased serum alanine aminotransferase in males
5. Increased bilirubin in males
6. Increased sodium, potassium, and calcium in males
7. Enlarged liver (possibly adaptive rather than adverse)
8. Increased kidney weight in males (absolute and relative)
9. Increased uterus weight in females (absolute and relative)
10. Increased incidence of Grade 2 hepatocyte hypertrophy in males and females.

Individually, none of the listed effects stand out as remarkable. However, collectively they could be considered as adverse and demonstrate that exposure to the high dose (500 mg/kg bw/day) of the test item is not well tolerated and/or such effects were not observed in the low (50 mg/kg bw/day) and middle (150 mg/kg bw/day) dose groups.

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)