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REACH

2-methylpentyl 2-hydroxybenzoate

EC number: 850-366-8 | CAS number: 98969-19-8

Life Cycle description
Uses advised against

Toxicological information

Repeated dose toxicity: oral

Administrative data

Endpoint:: short-term repeated dose toxicity: oral
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 29-07-2020 to 20-04-2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study
Remarks:: Guideline study performed under GLP. All relevant validity criteria were met.

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-07-2020 to 20-04-2021

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:

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:

GLP compliance:

yes

Limit test:

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) Pre-diet formulation: In refrigerator (2-8°C) protected from light container flushed with nitrogen ; (ii) Formulated-diet: Diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Diet was allowed to thaw before feeding commenced. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Stability under test conditions:
(i) Pre-diet formulation: Stable
(ii) Formulated-diet: Stable (for up to 21 days when stored in a closed container at room temperature and/or when stored frozen at ≤ -15°C in a closed container); formulated-diet was prepared and maintained for 12 days until replaced with diet, that taken from stored diet or freshly prepared diet.
In a preceding dietary formulation and method validation and/or within the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report). Specifically, within the preceding in a dietary formulation and method validation : stability for at least 12 days in an open container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in a closed container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in the freezer (≤ -15°C) in a closed container was demonstrated, over the concentration range 500 to 15000 ppm (full details available in the full study report). Concentration/homogeneity analysis was performed within the definitive study: Duplicate sets of samples (approximately 5 g) were used for concentration/homogeneity analysis, the remaining samples were retained at the testing facility as backup samples. Concentration/homogeneity results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Dietary analyses, during the definitive test confirmed that test item containing diets were prepared accurately and
homogenously.
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.
- 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: No. The test item was directly prepared into formulated diet. By the following:
The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Standard powder rodent diet (certified diet, recognised supplier) was utilized Diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Preliminary purification step (if any): Not applicable.
- Preparation of a nanomaterial dispersion (incl. dilution): 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): Not applicable. Applied in formulated diet.

INFORMATION ON NANOMATERIALS
- Chemical Composition: Not applicable.
- Density: Not applicable.
- Particle size & distribution: Not applicable.
- Specific surface area: Not applicable.
- Isoelectric point: Not applicable.
- Dissolution (rate): Not applicable.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not applicable.
- other information: 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 relevant OECD TG 422 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: 287 – 340 g; Females: 196 – 242 g
- Fasting period before study: No.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages (Macrolon MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage housed up to 5 per cage (see above, Macrolon MIV type height 18 cm). Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles. Group housed males and females and individual housed females, including females during mating, gestation and with litters, were housed in plastic cages containing appropriate bedding and according to relevant legislative requirements. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without environmental enrichment, bedding material, food and water.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: seven days before commencement of treatment. Females: seven days between arrival and start of estrous cycle smears (females) i.e. seven days before start of the pretest period

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified powdered diet, ad libitum – batch numbers and certificates of analysis citations were 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 24°C)
- Humidity (%): 55 ± 15 (or 40 to 70 : actual 48 to 73% ; The values that were outside the targeted range occurred for 8 days with a maximum of 73% and were without a noticeable effect on the clinical condition of the animals, and it was considered without noticeable effect on the outcome of the study).
- Air changes (per hr): > 10 per hour (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2020-07-29 To: 2020-10-08

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): equivalent to every 12 days ; specifically: diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Diet was allowed to thaw before feeding commenced. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z ; certified diet, recognised supplier)
- Storage temperature of food: room temperature and/or for stored diet (for replacement) : room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use. Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable
(ii) Formulated-diet: Stable (for up to 21 days when stored in a closed container at room temperature and/or when stored frozen at ≤ -15°C in a closed container); formulated-diet was prepared and maintained for 12 days until replaced with diet, that taken from stored diet or freshly prepared diet.
In a preceding dietary formulation and method validation and/or within the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report). Specifically, within the preceding in a dietary formulation and method validation : stability for at least 12 days in an open container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in a closed container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in the freezer (≤ -15°C) in a closed container was demonstrated, over the concentration range 500 to 15000 ppm (full details available in the full study report). Concentration/homogeneity analysis was performed within the definitive study: Duplicate sets of samples (approximately 5 g) were used for concentration/homogeneity analysis, the remaining samples were retained at the testing facility as backup samples. Concentration/homogeneity results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Dietary analyses, during the definitive test confirmed that test item containing diets were prepared accurately and
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.

Details on mating procedure:

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- Diet formulations and/or test item administration was examined in a preceding: dietary formulation and method validation and/or within the definitive test (full details available in the full study report).
- The formulated diet analysis consisted of UPLC-TUV analysis with external calibration. The method was calibrated by using calibration standards of the test item response between six nominal concentrations of 20 mg/L and 120 mg/L (working solutions), that were prepared in acetonitrile from two stock solutions (2000 mg/L) within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis in duplicate by UPLC-TUV. The analytical method was validated (details available within the full study report). For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99.
- Quality Control (QC) samples: Approximately 5 g blank powder diet was spiked with the test item at a target concentration of 1500 or 7500 ppm (using a spiking solution at 4000 mg/L concentration in acetonitrile). After spiking, the test item was allowed to adsorb to the diet for at least 30 minutes. The QC samples were treated similarly as the study samples. mean accuracies of the QC samples were within the criterion range of 80-120%. It demonstrated that the analytical method was adequate for the determination of the test item in the study samples. Actual mean accuracy in target concentrations were : 1500 ppm: 95% and/or 7500 ppm: 98%, respectively.
- Stability: The test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report).
- Study samples: Samples of approximately 60 g or 250 g were taken from the diets. For determination of accuracy, samples were taken at the random position or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the diets. Diet residue samples were taken from a blank diet which was prepared after routine cleaning of the diet preparation equipment. Duplicate samples of approximately 5 g of the powder diets were accurately weighed into glass vessels. The samples were extracted at 225 rpm with 50 mL acetonitrile. The shaking time was 30 minutes. The solutions were filtered through a 0.2 μm Spartan 30/0.2 RC filter and analysed. If necessary, the samples were further diluted with acetonitrile to obtain concentrations within the calibration range (dilution factors, where applicable reported in the full study report). In the Group 1 (control ; 0 ppm) diets, no test item was detected. The concentrations analysed in the diets of Group 2 (1500 ppm), Group 3 (3750 ppm) and Group 4 (7500 ppm) were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). Actual: group 2, 3 and 4 accuracies : 92%, 93% and 93%, respectively. The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Actual group 2 and 4 homogeneity CoV : 0.68% and 0.66%, respectively.

Duration of treatment / exposure:

The route of administration was in accordance with the relevant OECD TG 422 guideline.
The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards.
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. 57 days and females that failed to deliver: 42 to 44 days and/or total litter loss: 41 to 42 days.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the pre-mating, mating and post-coitum period. During the lactation period test item concentrations in the diet were adjusted based on historical control data for relative food consumption. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.

Frequency of treatment:

Continuous via diet
F1 generation were not dosed (were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper).

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet

Dose / conc.:

1 500 ppm

Remarks:

Low – Group 2

Dose / conc.:

3 750 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

7 500 ppm

Remarks:

High – Group 4

Dose / conc.:

99 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

501 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 7500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

109 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for females for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

275 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for females for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

534 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for females for 7500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)
note: data of only one female was present during the lactation period (mean intake 281 mg/kg bw/day) and therefore this period was not included in the Mean of means calculation.

No. of animals per sex per dose:

10 per sex per dose (10 male / 10 female) with satelliete 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/feces, or spilled diet from the food hopper).

Males:
Control (toxicity test) = 10
Control (recovery phase) = 5
1500 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Females:
Control (toxicity test) = 10
Control (recovery period) = 5
1500 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: The dose levels selected for investigation in this study (0, 1500, 3750 and 7500 ppm) by dietary administration were chosen based upon the results obtained in a 14-day dose range finding study (full details available in the full study report). Samples for diet analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed. However, to limit the impact, the diet preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 12 days after preparation of the diets. Homogeneity and stability of the diet under test conditions was demonstrated in the analytical method development and validation study (cited in the full study report ; also used within the definitive study where concentration analysis was performed). Dose levels in the 14-day DRF test were: Group 1: 7500 ppm and Group 2: 15000 ppm ; administered by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 10 days. Based upon the results of the acute oral toxicity test (anon., 2020) and/or considering the structural class of the substance, staggered start approach was chosen for this dose range finder study for ethical reasons. The study was initiated with Group 1. At 7500 ppm no clinical signs were observed. Food consumption during Week 1 of administration (especially Days 1-4) was very low, causing a significant body weight loss in 3/3 females. During Week 2 of administration food consumption completely recovered to normal levels, and body weights recovered to levels just below the historical control (HCD) range. Based on this data, 15000 ppm was selected as dose level for Group 2. At 7500 ppm: there was no mortality, no significant clinical findings were apparent. There was severe body weight loss over Days 1-4 of treatment, which recovered to start weight on Day 8-11. Body weight remained slightly low at the end of treatment. Food consumption was severely low on Days 1-4, followed by recovery to normal values from Days 8-11 onwards. There was no macroscopic abnormalities noted. Liver and kidney weights were considered to be normal. The mean test item intake was considered to be 490 mg/kg bw/day (at 7500 ppm nominal). At 15000 ppm: 3/3 females were sacrificed in extremis on Day 11. Within clinical signs, there was hunched posture and piloerection starting at Day 6-7 in 3/3 females. There was severe body weight loss from Day 4 of treatment onwards and/or food consumption was slightly low over Days 1-4 (actual value lower due to food spillage); severely low from Day 4-8, followed by partial recovery from Days 8-11. There was no macroscopic abnormalities noted. Organ weights (kidney and liver weights) were stated as ‘not available’ (not determined since terminated at in extremis not at ‘scheduled necropsy’). The mean test item intake was considered to be 810 mg/kg bw/day (at 15000 ppm nominal).
- 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. Deviation: Recovery Males were not fasted before clinical pathology blood collection at the end of treatment. It was considered within the study: Clinical Pathology values were generally within the same range as for Main males (except for glucose levels). As concurrent control Recovery Males were also not fasted, sufficient evaluation could be performed. It was considered this was not a significant deviation.
- 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:

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 (Main and Recovery) 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. Since suspected treatment related effects were obtained in the first 14 days of treatment, daily vaginal lavage was performed for Recovery females during the treatment-free recovery period to investigate reversibility of effect.
- 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 terminated 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. 57 days and females that failed to deliver: 42 to 44 days and/or total litter loss: 41 to 42 days.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Cranial cavity and all orifices. With special attention to reproductive organs.
- organs weighed: 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:

no effects observed

Description (incidence and severity):

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

Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain, housed and treated under the conditions in the study. There were no apparent dose-related trends and at the incidence observed, these were considered to be unrelated to treatment with the test item.

For the purposes of reporting : For males, “Repro period” represented the mating phase. For females, “Repro period” represented the mating, post-coitum and lactation phase.

Mortality:

no mortality observed

Description (incidence):

There were no treatment related mortalities.

Two females of the 1500 ppm group (female #70 and #72) and two females of the 7500 ppm group (female #86 and #87) were euthanized on Lactation Day 1, as they had a total litter loss.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: treatment-related changes in body weight and body weight gain were observed in males and females were observed.

In males, body weight stasis was observed in the first week of treatment, followed by a lower body weight gain when compared to concurrent controls in the following 3 weeks of treatment. At the end of the treatment period, mean body weight was x0.88 of control. In the subsequent treatment-free period of 14 days, mean body weight recovered to x0.93 of control. Considering the magnitude of the changes, these findings were considered adverse.

In Main females lower body weight gain was noted when compared to concurrent controls from Day 4 post-coitum onwards, resulting in a mean body weight of x0.81 of control at the end of the gestation period. Possible test item-related effects on body weight during lactation could not be assessed since only one lactating female with a single pup was available in the 7500 ppm group. In Recovery females, body weight gain was decreased on Days 15 and 22 of the repro period, resulting in decreased body weights (up to x0.93 of control on Day 15 of the repro period). At the end of the treatment period and the 14-days treatment-free recovery period, mean body weight was respectively 96% and 97% of control. As body weights of Recovery females recovered and considering the magnitude of change, these were not considered toxicologically relevant.

At up to 3750 ppm: body weight and body weight gain were considered unaffected by treatment in males and females.

At 3750 ppm: in males, mean body weight gain was decreased on Days 1 and 8 of the mating period, which resulted in slightly decreased absolute body weights (up to x0.95 of control on Day 8 of the mating period, not statistically significant). In view of the slight nature of this change, it was regarded not to be toxicologically relevant. At 1500 ppm and 3750 ppm, in females, decreased body weight gain was recorded on respectively Day 20 and Day 17 post-coitum, resulting in slightly decreased mean body weights at the end of gestation (x0.93 and x0.94 of control, respectively). In absence of a dose-related trend, no toxicological relevance was attached to this finding. No treatment-related changes were recorded during the lactation period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: in both males and females, food consumption was slightly decreased during the first week of treatment (not statistically significant) which subsequently recovered.

In males, a trend towards decreased absolute food consumption was recorded throughout the treatment period (up to x0.84 of control during the first week of mating). In females, decreased absolute and relative food consumption was recorded on several days during the post-coitum period (up to x0.78 of control for absolute food consumption). This was considered treatment-related. The lower food consumption in females during the first week of treatment and over post-coitum Days 0-4 was transient and considered likely to be palatability-related and therefore, no toxicological relevance was attached to it.

Note that data of only one female was present during the lactation period and therefore food consumption could not be assessed.

In Recovery males and (nulliparous) Recovery females, food consumption before or after correction for body weight was similar to the control level over the repro period and in the treatment-free recovery period thereafter.

Food efficiency:

no effects observed

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:

no effects observed

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 male (#35) at 3750 ppm and/or one female #52 in the control group, after treatment and/or one male #13 in the recovery control group, only post termination.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects (within assessable groups).

At 7500 ppm: except for one lactating female (#90), no matching controls were available for Main females due to their different physiological status (two with total litter loss, two with implantation sites only) and shorter (about one week) treatment period. Therefore no summary tables for Group 4 Main females were available and toxicological evaluation at 7500 ppm was performed for (nulliparous) Recovery females only.

No toxicologically relevant changes were noted in haematological parameters in males up to 7500 ppm, (lactating) Main females up to 3750 ppm and (nulliparous) Recovery females at 7500 ppm.

Statistically significant changes in white blood cell (WBC), lymphocyte and large unstained cell (LUC) counts in Main male rats at 1500 and/or 3750 ppm were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Statistically significant changes in WBC, lymphocyte, monocyte and LUC counts in Recovery male rats and red blood cell (RBC) counts in Recovery female rats were considered to be unrelated to treatment with the test item as these occurred at the end of the recovery period only.

At up to 7500 ppm, for coagulation parameters: treated male rats and (nulliparous) Recovery females and/or (lactating) Main females up to 3750 ppm were considered not to have been affected by treatment. See notes above, (lactating) Main females at 7500 ppm could not be assessed due to absence of equivalent controls.

At 7500 ppm, statistically significantly longer prothrombin time (PT) in males at 7500 ppm was considered the result of a slightly low control mean and therefore unrelated to treatment with the test item. [Historic control data: F0-males (period 2017-2019): PT (s): mean = 18.2; P5 – P95 = 16.30-20.70 (n=321)].

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant clinical chemistry changes distinguished treated from control animals at the end of the treatment period. No changes were recorded after the 14-day treatment-free period. There was evidence of recovery.

At 7500 ppm: except for one lactating female (#90), no matching controls were available for Main females due to their different physiological status (two with total litter loss, two with implantation sites only) and shorter (about one week) treatment period. Therefore no summary tables for Group 4 Main females were available and toxicological evaluation at 7500 ppm was performed for (nulliparous) Recovery females only.

At up to 7500 ppm: in males, decreased total bilirubin concentrations starting at 1500 ppm and up to x0.61 of control at 7500 ppm, was observed. Within Recovery females at 7500 ppm they were x0.68x of control at the end of treatment. In males at 1500 and 3750 ppm this was considered the result of slightly high control values. [Historic Control Data: F0-males (period 2017-2019): Total bilirubin (umol/L): mean = 2.3; P5 – P95 = 1.60-3.10 (n=355)]. In males and females at 7500 ppm this change was regarded treatment-related.

At 7500 ppm: in males, Increased creatinine concentration x1.21x of control was observed, which was considered treatment-related. The decreased cholesterol concentration in males x0.76x of control was considered not toxicologically relevant based on the direction of this change.

Decreased potassium concentrations in females at 1500 and 3750 ppm were considered not toxicologically relevant, as the result of a slightly high control mean. [Historic Control Data: F0-females (period 2017-2019): Potassium (mmol/L): mean = 4.54; P5 – P95 = 3.410-5.320 (n=325)].

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. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

At 3750 ppm, in females, increased total movements and ambulations were recorded (x1.33 and x1.44 of control, respectively). As mean values remained within the historical control range no toxicological relevance was attached to this finding. Note that no matching controls were available for Group 4 females due to their different physiological state (i.e. failed to deliver) and shorter (about one week) treatment period. Motor activity in Group 4 and (nulliparous) Recovery females was considered within the normal range for rats of this age and strain.

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 7500 ppm: In males, test item-related microscopic findings after treatment were noted in the epididymides, testes, thymus and bone marrow. Degeneration of germ cells of tubules (mainly round spermatids) and spermatid retention was noted at 7500 ppm in the testes of a single Main male (#39), both findings slight, as well as moderate luminal cellular debris of the epididymides. Decreased lymphoid cellularity in the thymus was noted at a minimal degree in Main males. An increased incidence and severity of increased adipocytes in the bone marrow (sternum and femur) was noted in Main males up to slight degree. All of the listed test item-related findings of 7500 ppm Main males were absent in 7500 ppm Recovery males. For further information see table 3.

In females, test item-related microscopic findings after treatment were noted in the liver, spleen, kidneys, bone and bone marrow of the Main females and/or Recovery females. An increased incidence and severity of extramedullary haematopoiesis of the spleen was noted in Main females. In the female with the highest severity (#88, marked degree) extramedullary haematopoiesis was also noted in the liver at a slight degree. The extramedullary haematopoiesis in the Recovery females (minimal degree) was within background range values. Moderate degeneration and anatomically related slight dilation of tubules was noted in the kidneys of a single 7500 ppm Main female (#86). The degeneration was characterized by basophilia and sloughing of necrotic cells into the tubular lumen. Increased adipocytes (sternum and/or femur) and/or increased hematopoietic cellularity was noted in the bone marrow of Main females and was absent in Recovery females. Increased bone (femur, up to slight degree) was noted in both Main females and in Recovery females. There was an increase in trabecular bone, particularly below the growth plate at the metaphyseal side. For further information see table 4.

Possible test item-related microscopic findings were present in the female reproductive organs and mammary gland of 7500 ppm Main females and are described in the reproductive performance/reproduction sections. Findings of note: Abnormal estrous cycles (including inactive uterus epithelium and/or increased mucification of the vaginal epithelium) were noted in one Recovery female of the control group (#61) and in one Recovery female (#100). This was considered to be related to the frequent lavage procedure used in the Recovery females. For further information see table 5. and/or relevant reproduction/developmental toxicity sections.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Overall, it was considered that:
At 7500 ppm, increased bone (femur) was noted in Main and Recovery females. This finding alone, was at the recorded minimal or slight degree considered as non-adverse. Relation to the abnormal estrus cycle and/or poor reproductive performance noted in the current study could not be established. The bone marrow (sternum and/or femur) of Main males and females at showed a subtle increase in incidence and severity of increased adipocytes (up slight degree). A decreased lymphoid cellularity (minimal) of the thymus, correlating with a lower organ weight, was noted in Main males. These findings of the bone marrow and thymus were most likely related to a test item-related effect on the final body weight. Finally, a possible higher liver weight, without microscopic correlate, was observed in Main males. The findings of the bone marrow, thymus and liver, were considered non-adverse and showed full recovery after a 14-day treatment-free period (Kerlin et al., 2016; Palazzi et al., 2016).

For Main females at 7500 ppm, no matching controls were available for due to their different physiological status and shorter treatment period, and therefore no toxicological evaluation could not be performed.

No toxicologically significant effects were observed at up to 3750 ppm, however it was considered that within reproductive parameters, that further investigation was required.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

1. Thyroid Hormone Assessment:
(i) F0 generation :
1. Serum levels of T4 in F0-males were dose dependently lower by x0.60 and x0.19 of control (achieving statistical significance) at 3750 ppm and 7500 ppm, respectively. In Recovery males, no difference was recorded after the 14-day recovery period. There was evidence of recovery.
2. Serum levels of T4 in F0-females, at 7500 ppm were lowered at x0.55 of control (achieving statistical significance). In Recovery females at the end of treatment they were x0.23 of control (achieving statistical significance), which was considered treatment related. In Recovery females, increased T4 serum levels were recorded x1.52 of control (achieving statistical significance), after the end of the 14-day recovery period.
3. TSH levels were decreased in F0-males at all dose levels x0.81, x0.70 and x0.81 of control in Groups 2, 3 and 4 (or 1500, 3750 and 7500 ppm, respectively). At 7500 ppm, in nulliparous Recovery females they were x0.72 of control, while at 3750 ppm increased TSH in Main F0-females was recorded x1.83 of control. After a 14-day treatment-free period TSH levels were increased in Recovery males at x1.72 of control, while decreased in Recovery females x0.77 of control.

Within the study there was no correlates to the Thyroid in gross pathology, organ weights or histopathology. Consequently, T3 was not further analysed.

In this OECD TG 422 study, marked changes of total T4 and TSH were observed in low dose groups (males only) and in mid and high dose groups (both sexes). However, under the conditions of this screening study the toxicological relevance is not clear, and no adverse effect or other correlates were observed that could be linked to these changes. Therefore these were not taken into account when determining the parental NOAEL.

Since the reproduction and developmental NOAEL could not be established within this study, as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation. The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test (OECD TG 421) and were determined to be at least 3750 ppm. T4 and/or TSH were further investigated therein.

(ii) F1 offspring:
Serum T4 levels in PND 14-16 males and females were considered to be unaffected by treatment.

At 1500 and 3750 ppm, slightly increased serum T4 levels in female pups up to x1.16 of control were considered not related to treatment with the test item considering the magnitude of the change (≤ 20%).

In the related gross pathology/histopathology:

The thyroids of the F1 offspring were considered unaffected by treatment up to 3750 ppm.

There were no other macroscopic findings finding observed in the offspring that were attributable to parental treatment with the test item up to 3750 ppm.

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: estrous cycle regularity was affected in 7/10 Main females and in 3/5 Recovery females, being irregular or acyclic. This was considered treatment-related. After a 14-day treatment-free period, estrous cycle regularity was recovered to regular in 2/3 Recovery females (both acyclic during the treatment period). For the Recovery female at 7500 ppm with irregular cycle during treatment, cycle regularity in the treatment-free recovery period could not be determined as it had only one complete estrous cycle (of 4 days), followed by an extended period of diestrus. Note that in two Recovery females of the control group, estrous cycle was irregular or acyclic during the recovery period, in addition.

At up to 3750 ppm: length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item. All females had regular cycles of 4 to 5 days.

At 7500 ppm: gestation length and gestation index were affected by treatment. Only 3/10 pregnant females delivered. Two had total litter loss before first litter check. For the single female (#90) which had delivered a single live pup, a gestation period of 23 days was recorded compared to 21-22 days in all other females with a normal litter (including controls). The gestation indices were respectively: 100%, 78%, 100% and 10% for the control, 1500, 3750 and 7500 ppm groups, respectively.

At 1500 ppm: The gestation index of 78% at 1500 ppm is caused by the total litter loss on Lactation Day 1 of Female #70 and #72. This is considered to be treatment-related as a clear effect on gestation index is noted at 7500 ppm and the incidence of gestation index is below the range of historical control data range [F0-females (period 2016-2020): Gestation Index (%): mean = 98; P5 – P95 = 89-100 (n=145)].

The number of implantations (implantation sites) was considered affected by treatment at all dose levels. Decreased implantation sites were recorded in females at 7500 and 1500 ppm : at 10.1 and 10.6 (not statistically significant), respectively compared to 13.2 in the control group. At 3750 ppm, a trend towards decreased number of implantation sites was noted within the normal range of biological variation. Based on the magnitude of change at 1500 and 7500 ppm, this finding was considered test item-related and adverse.

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related. The live birth indices were 100, 94 and 99% for the control, 1500 and 3750 ppm groups, respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant ‘histopathology’ section). Female #70 delivered 5 dead pups which were severely cannibalized. Missing pups were considered to be cannibalized. Furthermore, at 3750 ppm one pup was found dead at first litter check with advanced autolysis and no milk in the stomach. No toxicological relevance was attributed to this dead pup as the incidence remained within the range considered normal for pups of this age.

The total number of offspring born compared to the total number of uterine implantations was considered to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94%, 83%, 88% and 1% for the control, 1500, 3750 and 7500 ppm groups, respectively.

See ‘reproductive performance’ for further information/discussion.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: a single Main male (#39) showed test-item related microscopic findings, including an abnormal spermatogenic cycle.

At 1500 ppm and 3750 ppm, testes examination revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: in females a test item-related failure in reproductive performance was noted. Only Female #90 produced healthy offspring. Two females (#86 and #87) showed total litter loss and the other seven females had implantation sites only. Major related microscopic findings were: (i) For the total litter loss: Vacuolation of corpora lutea of the ovaries (slight), presence of placental tissue (correlating in one female with black-brown hard contents, noted at necropsy), inflammation of the endometrium (moderate), presence of cellular and/or hemorrhagic debris of the uterus, increased mucification of the epithelium marked) and the presence of luminal debris (cellular, mucus and/or hemorrhagic) of the vagina and moderate lobuloalveolar developmental of the mammary gland. (ii) For the implantation sites only: Vacuolation of corpora lutea of the ovaries (minimal or slight) and the presence of implantation sites (sometimes with hemorrhages in it). The mammary glands of the selected females with implantation sites only (female #88 and #94) showed lobuloalveolar developmental, with vacuolation of the epithelium at minimal or slight degree. For further information see table 5.

In males: a single Main male (#39) showed test-item related microscopic findings, including an abnormal spermatogenic cycle.

At 3750 ppm and 1500 ppm: in females, one in each the control and 1500 ppm group and two females in the 3750 ppm group were not pregnant, as compared to an absence of not-pregnant females at 7500 ppm. Furthermore, two females at 1500 ppm showed total litter loss, with similar microscopic findings of the reproductive organs and mammary gland as the 7500 ppm females (described above).

In males, the testes of males at 1500 ppm and 3750 ppm revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

No abnormalities were seen in the reproductive organs of males and females which could account for the lack of (healthy) offspring at 0, 1500 and 3750 ppm, and in absence of a clear dose-response it was considered as unrelated to treatment with the test item.

There was no effect on fertility index or mating index (mating performance) related to treatment. Precoital time was unaffected by treatment.

Mating indices were 100%, 100%, 100% and 100% for the control, 1500, 3750 and 7500 ppm groups, respectively.

Fertility indices were 90%, 90%, 80% and 100% for the control, 1500, 3750 and 7500 ppm groups, respectively.

All females showed evidence of mating.

The number of implantations (implantation sites) was considered affected by treatment at all dose levels. Decreased implantation sites were recorded in females at 7500 and 1500 ppm : at 10.1 and 10.6 (not statistically significant), respectively compared to 13.2 in the control group. At 3750 ppm, a trend towards decreased number of implantation sites was noted within the normal range of biological variation. Based on the magnitude of change at 1500 and 7500 ppm, this finding was considered test item-related and adverse.

Overall, it was considered that:
At 7500 ppm: There was an adverse test-item related effect in the reproductive performance of 7500 ppm. At lower dose levels, there were no clear dose response relationship(s) apparent and no consistency in animal response(s) across all dose levels. Furthermore, histopathological examination of the male and female reproductive organs revealed no clear causal relationship(s). At 7500 ppm, the observed findings of the ovaries, uterus, vagina, and mammary gland in Main females at were in concordance with physiological status (i.e. total litter loss shortly after delivery or fetus loss during pregnancy) and secondary to the failure in reproductive performance, but with no clear causal relationship for the lack of healthy offspring. The increase in incidence and/or severity of extramedullary haematopoiesis of the spleen (correlating with enlargement) and liver, as well as the increased hematopoietic cellularity of the bone marrow were also considered to be related to this failure in fulfilling a normal pregnancy. These findings are regarded to be related to a response on blood loss. Tubular degeneration and dilation of the kidney was noted at 7500 ppm in a single Main female, which lost her litter shortly after delivery. Since these microscopic findings of the kidneys occurred at a single incidence, were present in the high dose group, and can sometimes be observed in females with total litter loss and poor health condition, they were regarded to be related to/correlated towards with her failure in reproductive performance.

It was considered that as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. Consequentially, no reproductive NOAEL could be established. This required further investigation.

In relation to Thyroid Hormone Assessment - see cross-referenced study OECD TG 421 (2021). Further comments are provided therein as part of applicant assessment with relevant literature citations.

Reference: 'Toxicity to reproduction - OECD 421 - 2021' - section: F0 and/or F1 'other effects' : Thyroid Hormone Assessment

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

>= 3 750 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

>= 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

>= 275 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Critical effects observed:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

At up to 3750 ppm: no toxicologically significant clinical signs occurred among offspring (F1) that were considered to be related to treatment with the test item.

Viability indices were 100, 99 and 98% for the control, 1500 and 3750 ppm groups, respectively. One pup at 1500 ppm with no milk in the stomach and two pups at 3750 ppm were found dead or missing on PND 2-4. The pup at 3750 ppm that was missing on PND 2 was cold to touch and presented with blue spots the day before. Most likely it had been cannibalized. These findings were considered unrelated to treatment and within the range considered normal for the species and strain, at this age.

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

At 7500 ppm (high dose group) : there was treatment related mortality. Only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

At 3750 ppm : there were no treatment related mortality associated with treatment within F1 males or females. This is considering that there was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, absence of adverse effects to nipple counts and/or macropathology findings in offspring.

Viability indices were 100, 99 and 98% for the control, 1500 and 3750 ppm groups, respectively. One pup at 1500 ppm with no milk in the stomach and two pups at 3750 ppm were found dead or missing on PND 2-4. The pup at 3750 ppm that was missing on PND 2 was cold to touch and presented with blue spots the day before. Most likely it had been cannibalized. These findings were considered unrelated to treatment and within the range considered normal for the species and strain, at this age.

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

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related.

The live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 100, 94 and 99% for the control, 1500 and 3750 ppm groups, respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant F0 ‘histopathology’ section).

It was considered that as test item related developmental effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level and that consequentially, no reproductive NOAEL could be established. Further investigation was required.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

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

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

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:

no effects observed

Description (incidence and severity):

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

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

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

At up to 3750 ppm: Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. Treatment had no effect on areola/nipple retention. No males at PND 13 had nipples.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

At up to 3750 ppm: Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. Treatment had no effect on areola/nipple retention. No males at PND 13 had nipples.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

At up to 3750 ppm: A check at PND13 was performed to assess for the presence or absence of nipple/areolae for the male offspring; no nipples were found.

Organ weight findings including organ / body weight ratios:

not examined

Description (incidence and severity):

There were no macroscopic findings or needed histopathological findings observed that were attributable to parental treatment with the test item. Consequently no organ weights were determined within F1 males or females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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):

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Table 1.0 : Test Item Intake within the Dietary Study

		Mean over means intake [mg test item/kg body weight] (mean range indicated in bracktes)
	Group Number	2		3		4
	Nominal dietary inclusion level (ppm)	1500		3750		7500
Sex	Study Period
Male	Pre-mating	103	(102-105)	261	(258-264)	512	(473-551)
	Post-mating	95	(93-98)	238	(227-250)	489	(481-497)
	Mean of means #a	99		250		501
Female	Pre-mating	109	(108-110)	261	(254-268)	555	(459-650)
	Post-coitum	111	(102-121)	278	(263-289)	519	(452-565)
	Repro period (Recovery females)	n.a.		n.a.		480	(447-516)
	Lactation	104	(95-112)	211	(274-296)	281 #b	(246-317)
	Mean of means #a	109		275		534

where:

#a : Mean of means of all periods, weighed for number of measurement days per period:

Males: ((14 x mean premating) + (14 x mean mating)) / 28

Females: ((14 x mean premating) + (20 x mean post-coitum) + (12 x mean lactation)) / 46

#b : Data of only one female was present during the lactation period and therefore this period was not included in the Mean of means calculation.

For Main males, “Repro period” represents the mating phase.

For Main females, “Repro period” represents the mating, post-coitum and lactation phase.

For Recovery animals, the “Premating period” and “Repro period” represents the treatment phase.

Notes:

The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the pre-mating, mating and post-coitum period. During the lactation period test item concentrations in the diet were adjusted based on historical control data for relative food consumption (documented in the full study report).

Test item intake was calculated as concentration of test item in diet (ppm) against relative food consumption.

Table 2.0 : Mean Percent Thymus and Liver Weight Differences from Control Groups (F0 males/females)

		Main males			Recovery males
Dose level (ppm)		1500	3750	7500	7500
THYMUS	Absolute	-16	9	-36	-1
	Relative to B.W.	-9	17	-27	6
LIVER	Absolute	-3	-4	-3	-9
	Relative to B.W.	4	3	12 **	-3

Where:

B.W. : body weight

** : P < 0.01

Table 3.0 : Summary Test Item-Related Microscopic Findings – Scheduled Termination F0 Males.

	Main males				Recovery males
Dose level (ppm)	0	1500	3750	7500	0	7500
EPIDIDYMIDES #a	6	5	6	5	5	5
Cell debris
Moderate	-	-	-	1	-	-

TESTES #a	6	5	6	5	5	5
Spermatid retention
Slight	-	-	-	1	-	-
Degeneration tubule
Slight	-	-	-	1	-	-

THYMUS #a	5	5	5	5	5	5
Decreased cellularity
Minimal	-	-	-	2	-	-

BONE MARROW, STERNUM #a	5	5	5	5	4	5
Increased adipocytes
Minimal	-	-	-	1	-	-
Slight	-	-	-	1	-	-

BONE MARROW, FEMUR #a	5	5	5	5	5	5
Increased adipocytes
Minimal	1	-	1	1	1	-
Slight	-	-	-	1	-	-

#a : number of tissues examined from each group

Table 4.0 : Summary Test Item-Related Microscopic Findings – Scheduled Termination F0 Females.

	Main females				Recovery females
Dose level (ppm)	0	1500	3750	7500	0	7500
LIVER #a	5	5	5	5	5	5
Extramedullary haematopoiesis
Slight	-	-	-	1	-	-

SPLEEN #a	5	5	5	5	5	5
Extramedullary haematopoiesis
Minimal	3	2	5	1	2	3
Slight	1	-	-	1	-	-
Moderate	-	-	-	1	-	-
Marked	-	-	-	1	-	-

KIDNEYS #a	5	5	5	5	5	5
Degeneration tubule
Moderate	-	-	-	1	-	-
Dilation tubule
Slight	-	-	-	1	-	-

BONE MARROW, STERNUM #a	5	5	5	5	5	5
Increased adipocytes
Minimal	-	-	-	3	1	-
Slight	-	-	-	1	-	-
Increased cellularity
Minimal	-	-	-	1	-	-
Slight	-	-	-	1	-	-

BONE MARROW, FEMUR #a	5	5	5	5	5	5
Increased adipocytes
Minimal	-	-	-	1	1	-
Slight	-	-	-	-	-	-
Increased cellularity
Minimal	-	-	-	-	-	-
Slight	-	-	-	1	-	-

BONE, FEMUR #a	5	5	5	5	5	5
Increased bone
Minimal	-	-	-	3	-	3
Slight	-	-	-	2	-	1

#a : number of tissues examined from each group

Table 5.0 : Summary of In-Life Reason for F0 Males that Failed to Sire and F0 Females that Failed to Deliver Healthy Pups.

Group	Dose level (ppm)	Female/Male number	In-life Reason
1	0	58 / 08	Not pregnant
2	1500	68 / 18 70 / - 72 / -	Not pregnant Total litter loss Total litter loss
3	3750	76 / 26 82 / 32	Not pregnant Not pregnant
4	7500	86 / - 87 / - 88 / - 89 / - 91 / - 92 / - 93 / - 94 / - 95 / -	Total litter loss Total litter loss Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only

Conclusions:

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) for systemic toxicity was considered to be ≥ 3750 ppm (equivalent to 250 mg/kg bw/day in males and/or 275 mg/kg bw/day in females) due to decreased body weights and clinical biochemistry changes in males at 7500 ppm. For reproductive and developmental toxicity, the NOAEL for males and females the NOAEL could not be established as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation.
Note: The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test, according to OECD TG 421. There general toxicity and reproductive/developmental toxicity NOAELs were determined to be ≥ 3750 ppm.

Executive summary:

The study was performed according the requirements of OECD TG 422 guideline under GLP conditions. Following a previously conducted 10-day sighting study, the systemic toxic potential of the test item in Wistar Han 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 with additional subgroups used to assess reversibility, persistence or delayed effects for 14 days post treatment. Three toxicity treatment groups with a control were conducted, each comprising ten male and ten female rats which received dietary test item at doses of 0 (Control), 1500, 3750 or 7500 ppm test item (Group 1 to 4, respectively). Satellite recovery phase groups included five males and females treated at 0 ppm (Control) and 7500 ppm. During the lactation period, the following dietary concentrations were used based on historical control data for relative food consumption: days 1-4: Group 1: 0 ppm, Group 2: 1000 ppm, Group 3: 2500 ppm and Group 4: 5000 ppm ; days 4-7: Group 1: 0 ppm, Group 2: 750 ppm, Group 3: 1875 ppm and Group 4: 3750 ppm; day 7 onwards: Group 1: 0 ppm, Group 2: 600 ppm, Group 3: 1500 ppm and Group 4: 3000 ppm. Chemical analyses of dietary preparations were conducted once during the study to assess accuracy and homogeneity. A similarly constituted Control group received, untreated (no test item) diet, throughout the same relative treatment period. Overall mean achieved doses at 1500, 3750 or 7500 ppm for the toxicity and recovery phase were 99, 250 and 501 mg/kg bw/day in males and 109, 275 and 534 mg/kg bw/day in females, respectively. The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 28 days. Main males and Recovery males were exposed for 29-30 days, including a minimum of 14 days prior to mating and the duration of the mating period for Main males. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females were exposed for 51-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and at least 14 days after delivery, up to and including the day of scheduled necropsy. Main females that failed to deliver pups were treated for 42-44 days. Main females which had a total litter loss were treated for 41-42 days. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females (57 days). The offspring received no direct administration of the test item; any exposure was in utero or via the milk or inadvertent exposure from maternal urine/faeces or spilled diet from foot hoppers. 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 hormones T4 and TSH (F0-males and F0-females), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, sex, anogenital distance, areola/nipple retention and macroscopy, measurement of thyroid hormone T4 (PND 14-16 pups)). No treatment-related changes were noted in any of the following parameters investigated in this study, i.e. mortality, clinical appearance, functional observations (motor activity, grip strength, hearing ability, pupillary reflex and static righting reflex), haematology and coagulation parameters and macroscopic examination.

Toxicity phase results

At 7500 ppm toxicity phase (i.e. main) groups males and females, treatment-related changes in body weight and body weight gain were observed, accompanied by decreased food consumption. In main males mean body weight was 12% lower compared with control at the end of the treatment period. In the subsequent treatment-free period of 14 days, mean body weight recovered to being 7% lower than controls. Considering the magnitude of change, these findings were considered adverse effects. In toxicity phase (i.e. main) females, mean body weight gain was decreased from Day 4 post-coitum onwards, resulting in a mean body weight 19% lower than control at the end of the gestation period. The lower food consumption in females at 7500 ppm during the first week of treatment and over post-coitum Days 0-4 was transient and considered likely to be palatability-related and therefore, no toxicological significance. It was noted that potential test item-related effects on body weight and food consumption during lactation could not be assessed fully, since only one lactating female with a single pup was available in the 7500 ppm group. At 7500 ppm, clinical biochemistry parameters were affected by treatment. Including : decreased total bilirubin concentrations in males and nulliparous recovery females; increased creatinine concentrations in males at end of treatment. It was considered these findings were adverse. No matching controls were available for main females at 7500 ppm due to their different physiological statuses and shorter treatment periods. Toxicological evaluation was therefore not performed. Serum levels of total T4 were decreased at 3750 and 7500 ppm in F0-males, at 3750 ppm in toxicity phase (i.e. main) F0-females and at 7500 ppm in nulliparous recovery females at the end of treatment. In recovery males, no difference was recorded after a 14-day treatment-free period. In recovery females increased serum T4 levels were recorded after the 14-day treatment-free period. TSH levels were decreased in F0-males at all dose levels and at 7500 ppm in nulliparous recovery females, whereas increased at 3750 ppm main F0-females. After a 14-day treatment-free period TSH levels were increased in recovery males and decreased in recovery females. Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment. Test item-related microscopic findings were recorded in males and females at 7500 ppm: increased bone (femur) was noted in main and recovery females at 7500 ppm, recorded minimal or slight degree therefore considered as non-adverse. The bone marrow (sternum and/or femur) of main males and females at 7500 ppm showed a subtle increases in incidence and severity of increased adipocytes (slight degree). A decreased lymphoid cellularity (minimal) of the thymus, was seen in main males at 7500 ppm correlating with a lower organ weight. These findings of the bone marrow and thymus were considered to be most likely related to a test item-related effect on final body weights. A potential higher liver weight without microscopic correlates, was observed in main males at 7500 ppm. The findings of the bone marrow, thymus and liver, were considered non-adverse effects and which demonstrated full recovery after a 14-day treatment-free period. No matching controls were available for main females at 7500 ppm due to their different physiological statuses and shorter treatment periods. Toxicological evaluation was therefore not performed.

Reproductive results

At 7500 ppm, estrous cycle regularity was affected in 7/10 main and 3/5 recovery females, being irregular or acyclic. This was considered treatment-related at the incidence observed. After a 14-day treatment-free period, estrous cycle regularity was recovered to regular in 2/3 acyclic recovery females but not for the third female. There was an adverse test-item related effect in the reproductive performance of 7500 ppm main group females, resulting in a low number of mean implantation sites and only one female with healthy offspring. At 3750 ppm, a trend towards a decreased number of implantation sites was noted although remained within the normal range of biological variation. At 1500 ppm, a lower number of implantation sites in females was observed in 3/9 animals. Even though no clear dose response relationship was apparent and no consistency in response was seen across all doses, the results may reflect the potential for toxicity at lower dose, It was considered this would require further investigation to clarify. Histopathological examination of the male and female reproductive organs revealed no clear causal relationship. At 7500 ppm, a single main male showed an abnormal spermatogenic profile, recorded as degeneration of germ cells and spermatid retention of the testes and the presence of luminal cellular debris of the epididymides. These findings were regarded adverse in this specific male. However it was noted this was observed at isolated incidence, and all main Group 4 males had mated and no clear causal relationship was apparent for the lack of healthy offspring in these males. Within 7500 ppm recovery group males, this finding was absent. For main females at 7500 ppm, the observed findings of the ovaries, uterus, vagina, and mammary gland were in concordance with their physiological status (i.e. total litter loss shortly after delivery or fetus loss during pregnancy) and secondary to the failure in reproductive performance, but with no clear causal relationship for the lack of healthy offspring. The increase in incidence and/or severity of extramedullary haematopoiesis of the spleen (correlating with enlargement) and liver, as well as the increased hematopoietic cellularity of the bone marrow were also considered to be related to this failure in fulfilling a normal pregnancy. These findings are regarded to be related to a response on or to blood loss. Tubular degeneration and dilation of the kidney was noted in a single main female at 7500 ppm, which lost her litter shortly after delivery. Since these microscopic findings of the kidneys occurred at a single incidence, were present in the high dose group, and can sometimes be observed in females with total litter loss and poor health condition, they were regarded to be related with her failure in reproductive performance, in addition as well. No toxicologically significant changes were noted in any of the remaining reproductive parameters investigated in this study (i.e. mating and fertility indices and precoital time).

Developmental results

At 7500 ppm, a test item-related severe decrease in gestation index and post-implantation survival index was observed In this high dose group, only three females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed in the high dose group. At 1500 ppm, a test item-related decrease in gestation index, post-implantation survival index, live litter size and live birth index was observed. In this group, only 5/10 females produced a healthy normal litter (two females with a total litter loss, two females with a few pups only and one female not pregnant). At 7500 ppm, a clear effect was noted in females, whereas at 1500 ppm females developmental effects could not be disregarded. At up to 3750 ppm, no toxicologically significant changes were noted in any of the remaining developmental parameters investigated (i.e. viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopic examination).

Conclusion:

Under the conditions of this study, following dietary administration of test item to Han Wistar rats indicated the parental No-Observed-Adverse-Effect-Level (NOAEL) was considered to be 3750 ppm (or mean achieved doses; males 250 mg/kg bw/day and/or females: 275 mg/kg bw/day), based on decreased body weights, clinical biochemistry at higher dose levels. Marked changes in parental T4 and TSH was observed in lower (male only) and mid-level (both sexes) dose levels, however with no linked adverse effects. Toxicological relevance was unclear. No effects in offspring T4 was noted. For reproduction and developmental effects the No-Observed-Adverse-Effect-Level (NOAEL) could not be established.

Applicant assessment indicates: the reproduction and developmental NOAEL could not be established in the OECD TG 422 study, as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation. The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test (OECD TG 421) and were determined to be at least 3750 ppm.

Cross-reference 2

Reason / purpose for cross-reference:: reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29-07-2020 to 20-04-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

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:

GLP compliance:

yes

Limit test:

Specific details on test material used for the study:

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on test animals or test system and environmental conditions:

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Details on mating procedure:

Duration of treatment / exposure:

Frequency of treatment:

Continuous via diet
F1 generation were not dosed (were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper).

Duration of test:

- Age at study initiation: F0 Males: ca. 10 weeks (i.e. 10 – 11 weeks old) ; F0 Females: ca. 13 weeks (I.e. 13-14 weeks)
- Age at mating of the mated animals in the study: after 14 days treatment – cohabited for mating: F0 generation: Males: ca. 12 weeks ; Females: ca. 15 weeks
(i.e. after two weeks treatment)
- Age at termination: [based on ages at study initiation : [F0 Males: ca. 14 weeks old and F0 Females: that delivered ca. 21 to 22 weeks old and that failed to deliver: ca. 19 to 20 weeks old

Dose / conc.:

0 ppm

Remarks:

Control – Group 1; Basal diet

Dose / conc.:

1 500 ppm

Remarks:

Low – Group 2

Dose / conc.:

3 750 ppm

Remarks:

Intermediate – Group 3

Dose / conc.:

7 500 ppm

Remarks:

High – Group 4

Dose / conc.:

99 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

501 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for males for 7500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

109 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for females for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

275 mg/kg bw/day (actual dose received)

Remarks:

Overall Mean for females for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Dose / conc.:

534 mg/kg bw/day (actual dose received)

Remarks:

No. of animals per sex per dose:

Control animals:

yes, plain diet

Details on study design:

Maternal examinations:

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

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

BODY WEIGHT: Yes
- Time schedule for examinations: 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: Yes. Individual Food Consumption (g/animal/day), Relative Food Consumption (g/Kg body weight/day) and/or test item intake (mg substance /Kg body weight/day) were all determined. Mean daily consumption per animal (g/animal/day) was calculated for each phase,
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- 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.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # : Male animals: F0 Males: after minimum of 28 days ; i.e. minimum of 14 days prior to mating and during the mating period and up to termination/necropsy (i.e. after 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: after ca. 57 days and females that failed to deliver: after 42 to 44 days and/or total litter loss: after 41 to 42 days.
- Organs examined: 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.
For histopathology: the relevant 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.

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).

Ovaries and uterine content:

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

Fetal examinations:

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

Statistics:

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

Indices:

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

Historical control data:

Historical control data was available.

Clinical signs:

no effects observed

Description (incidence and severity):

Mortality:

no mortality observed

Description (incidence):

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: treatment-related changes in body weight and body weight gain were observed in males and females were observed.

in males, body weight stasis was observed in the first week of treatment, followed by a lower body weight gain when compared to concurrent controls in the following 3 weeks of treatment. At the end of the treatment period, mean body weight was x0.88 of control. In the subsequent treatment-free period of 14 days, mean body weight recovered to x0.93 of control. Considering the magnitude of the changes, these findings were considered adverse.

In Main females lower body weight gain was noted when compared to concurrent controls from Day 4 post-coitum onwards, resulting in a mean body weight of x0.81 of control at the end of the gestation period. Possible test item-related effects on body weight during lactation could not be assessed since only one lactating female with a single pup was available in the 7500 ppm group. In Recovery females, body weight gain was decreased on Days 15 and 22 of the repro period, resulting in decreased body weights (up to x0.93 of control on Day 15 of the repro period). At the end of the treatment period and the 14-days treatment-free recovery period, mean body weight was respectively 96% and 97% of control. As body weights of Recovery females recovered and considering the magnitude of change, these were not considered toxicologically relevant.

At up to 3750 ppm: body weight and body weight gain were considered unaffected by treatment in males and females.

At 3750 ppm: in males, mean body weight gain was decreased on Days 1 and 8 of the mating period, which resulted in slightly decreased absolute body weights (up to x0.95 of control on Day 8 of the mating period, not statistically significant). In view of the slight nature of this change, it was regarded not to be toxicologically relevant. At 1500 ppm and 3750 ppm, in females, decreased body weight gain was recorded on respectively Day 20 and Day 17 post-coitum, resulting in slightly decreased mean body weights at the end of gestation (x0.93 and x0.94 of control, respectively). In absence of a dose-related trend, no toxicological relevance was attached to this finding. No treatment-related changes were recorded during the lactation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food efficiency:

no effects observed

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:

no effects observed

Description (incidence and severity):

Haematological findings:

no effects observed

Description (incidence and severity):

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: In males, potential test item related lower thymus weights (absolute and relative to body weights) and higher liver weights (relative to body weight were noted). The liver weight findings achieved statistical significance. Furthermore some organ weight differences were statistically significant when compared to the control group weight: absolute testes and prostate gland weights and relative brain weight. However, they were considered to be the result of a test item-related effect on final body weight (0.86x of control). There was no test item-related effect on the final body weight and organ weights of the Recovery males.

In females, assessment could not be completed. Selected females of the 7500 ppm group had a shorter treatment duration and different physiological status (implantation sites only or total litter loss), compared to the selected females of the control group (except for Female #90). Therefore, no comparison of organ weight of the 7500 ppm Main females with the control group could be made and no toxicological evaluation could be performed.

At up to 3750 ppm: there were not test item related effects on organ weights of main female.

Any other organ weight differences, including those that reached statistical significance (relative to body weight of the liver of 7500 ppm Recovery females) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, lack of microscopic correlate and/or general overlap and variability in individual values.

For further information see table 2. and/or ‘histopathological findings’. It was considered the findings of the bone marrow and thymus were most likely related to a test item-related effect on the final body weight. In Main males at 7500 ppm, the possible higher liver weight was without microscopic correlate. Therefore, the findings of the bone marrow, thymus and liver, were considered non-adverse and showed full recovery after a 14-day treatment-free period (Kerlin et al., 2016; Palazzi et al., 2016).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: a test item-related macroscopic finding was noted in a single female of the Main group, consisting of an enlarged spleen. The microscopic correlate was extramedullary haematopoiesis (marked degree).

For further information see table 4. and/or ‘histopathological findings’.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

1. Thyroid Hormone Assessment:
(i) F0 generation :
1. Serum levels of T4 in F0-males were dose dependently lower by x0.60 and x0.19 of control (achieving statistical significance) at 3750 ppm and 7500 ppm, respectively. In Recovery males, no difference was recorded after the 14-day recovery period. There was evidence of recovery.
2. Serum levels of T4 in F0-females, at 7500 ppm were lowered at x0.55 of control (achieving statistical significance). In Recovery females at the end of treatment they were x0.23 of control (achieving statistical significance), which was considered treatment related. In Recovery females, increased T4 serum levels were recorded x1.52 of control (achieving statistical significance), after the end of the 14-day recovery period.
3. TSH levels were decreased in F0-males at all dose levels x0.81, x0.70 and x0.81 of control in Groups 2, 3 and 4 (or 1500, 3750 and 7500 ppm, respectively). At 7500 ppm, in nulliparous Recovery females they were x0.72 of control, while at 3750 ppm increased TSH in Main F0-females was recorded x1.83 of control. After a 14-day treatment-free period TSH levels were increased in Recovery males at x1.72 of control, while decreased in Recovery females x0.77 of control.

Within the study there was no correlates to the Thyroid in gross pathology, organ weights or histopathology. Consequently, T3 was not further analysed.

In this OECD TG 422 study, marked changes of total T4 and TSH were observed in low dose groups (males only) and in mid and high dose groups (both sexes). However, under the conditions of this screening study the toxicological relevance is not clear, and no adverse effect or other correlates were observed that could be linked to these changes. Therefore these were not taken into account when determining the parental NOAEL.

Since the reproduction and developmental NOAEL could not be established within this study, as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation. The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test (OECD TG 421) and were determined to be at least 3750 ppm. T4 and/or TSH were further investigated therein.

(ii) F1 offspring:
Serum T4 levels in PND 14-16 males and females were considered to be unaffected by treatment.

At 1500 and 3750 ppm, slightly increased serum T4 levels in female pups up to x1.16 of control were considered not related to treatment with the test item considering the magnitude
of the change (≤ 20%).

In the related gross pathology/histopathology:

The thyroids of the F1 offspring were considered unaffected by treatment up to 3750 ppm.

There were no other macroscopic findings finding observed in the offspring that were attributable to parental treatment with the test item up to 3750 ppm.

Details on results:

Number of abortions:

no effects observed

Description (incidence and severity):

No signs of abortion or premature birth were observed within the study. No deficiencies in maternal care were noted up to 3750 ppm. (As data was available of only 3 females in the high dose group, a toxicological evaluation in the high dose group could not be performed).

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered to be affected by treatment with the test item. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 94%, 83%, 88% and 1% for the control, 1500, 3750 and 7500 ppm groups, respectively.

At 7500 ppm: gestation length and gestation index were affected by treatment. Only 3/10 pregnant females delivered. Two had total litter loss before first litter check. For the single female (#90) which had delivered a single live pup, a gestation period of 23 days was recorded compared to 21-22 days in all other females with a normal litter (including controls). The gestation indices were respectively: 100%, 78%, 100% and 10% for the control, 1500, 3750 and 7500 ppm groups, respectively.

At 1500 ppm: The gestation index of 78% at 1500 ppm is caused by the total litter loss on Lactation Day 1 of Female #70 and #72. This is considered to be treatment-related as a clear effect on gestation index is noted at 7500 ppm and the incidence of gestation index is below the range of historical control data range [F0-females (period 2016-2020): Gestation Index (%): mean = 98; P5 – P95 = 89-100 (n=145)].

The number of implantations (implantation sites) was considered affected by treatment at all dose levels. Decreased implantation sites were recorded in females at 7500 and 1500 ppm : at 10.1 and 10.6 (not statistically significant), respectively compared to 13.2 in the control group. At 3750 ppm, a trend towards decreased number of implantation sites was noted within the normal range of biological variation. Based on the magnitude of change at 1500 and 7500 ppm, this finding was considered test item-related and adverse.

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

Only Female #90 produced healthy offspring. Two females (#86 and #87) showed total litter loss and the other seven females had implantation sites only. Major related microscopic findings were: (i) For the total litter loss: Vacuolation of corpora lutea of the ovaries (slight), presence of placental tissue (correlating in one female with black-brown hard contents, noted at necropsy), inflammation of the endometrium (moderate), presence of cellular and/or hemorrhagic debris of the uterus, increased mucification of the epithelium marked) and the presence of luminal debris (cellular, mucus and/or hemorrhagic) of the vagina and moderate lobuloalveolar developmental of the mammary gland. (ii) For the implantation sites only: Vacuolation of corpora lutea of the ovaries (minimal or slight) and the presence of implantation sites (sometimes with hemorrhages in it). The mammary glands of the selected females with implantation sites only (female #88 and #94) showed lobuloalveolar developmental, with vacuolation of the epithelium at minimal or slight degree. For further information see table 5.

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of
offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related.

The live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 100, 94 and 99% for the control, 1500 and 3750 ppm groups,respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant ‘histopathology’ section).

Dead fetuses:

effects observed, treatment-related

Description (incidence and severity):

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related. The live birth indices were 100, 94 and 99% for the control, 1500 and 3750 ppm groups, respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant ‘histopathology’ section).

Changes in pregnancy duration:

effects observed, treatment-related

Description (incidence and severity):

At 7500 ppm: gestation length and gestation index were affected by treatment. Only 3/10 pregnant females delivered. Two had total litter loss before first litter check. For the single female (#90) which had delivered a single live pup, a gestation period of 23 days was recorded compared to 21-22 days in all other females with a normal litter (including controls). The gestation indices were respectively: 100%, 78%, 100% and 10% for the control, 1500, 3750 and 7500 ppm groups, respectively.

At 1500 ppm: The gestation index of 78% at 1500 ppm is caused by the total litter loss on Lactation Day 1 of Female #70 and #72. This is considered to be treatment-related as a clear effect on gestation index is noted at 7500 ppm and the incidence of gestation index is below the range of historical control data range [F0-females (period 2016-2020): Gestation Index (%): mean = 98; P5 – P95 = 89-100 (n=145)].

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects were observed in the study. For reproductive/developmental parameters: it was considered that as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level that consequentially, no reproductive/developmental NOAEL could be established. This required further investigation.

Key result

Dose descriptor:

NOAEL

Remarks:

general systemic toxicity

Effect level:

>= 3 750 ppm

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical biochemistry

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive/developmental toxicity

Based on:

test mat.

Remarks on result:

not determinable

Key result

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Reduction in number of live offspring:

effects observed, treatment-related

Description (incidence and severity):

Offspring survival was affected by treatment.

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related.

The live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 100, 94 and 99% for the control, 1500 and 3750 ppm groups, respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant F0 ‘histopathology’ section).

At 3750 ppm : there were no treatment related mortality associated with treatment within F1 males or females. This is considering that there was no effect of parental treatment on offspring clinical signs, sex ratio, offspring body weight, survival, ano-genital distance, absence of adverse effects to nipple counts and/or macropathology findings in offspring.

Important note:
At 7500 ppm (high dose group) : Only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Live litter sizes were considered affected by treatment. Note that at 7500 ppm, female #90 had 1 pup only. A lower mean number of living pups was recorded at 1500 ppm 9.3 against, 12.4 in the control group, which was related to the lower number of implantation sites in this group. As a trend towards a lower mean number of living pups was also noted at 3750 ppm, this finding was considered to be treatment-related. The number of live offspring on Day 1 after littering compared to the total number of offspring born was lower at 1500 ppm compared to the concurrent control. As at 1500 ppm, two females had total litter loss, comparable to the findings noted at 7500 ppm, the lower live birth index was considered to be treatment-related.

The live birth indices (number of live offspring on PND 1 as percentage of total number of offspring born) were 100, 94 and 99% for the control, 1500 and 3750 ppm groups, respectively. At 1500 ppm, Female #70 and #72 and at 7500 ppm, Female #86 and #87 had total litter loss on Lactation Day 1 or before first litter check, all with similar microscopic findings of the reproductive organs and mammary gland (see relevant F0 ‘histopathology’ section).

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

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

At up to 3750 ppm: The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment with the test item.

Viability indices (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) were 100, 99 and 98% for the control, 1500 and 3750 ppm groups, respectively. One pup at 1500 ppm with no milk in the stomach and two pups at 3750 ppm were found dead or missing on PND 2-4. The pup at 3750 ppm that was missing on PND 2 was cold to touch and presented with blue spots the day before. Most likely it had been cannibalized. These findings were considered unrelated to treatment and within the range considered normal for the species and strain, at this age.

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

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment with the test item. The lactation indices were 100% for the control, 1500 and 3750 ppm groups, respectively.

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

External malformations:

no effects observed

Description (incidence and severity):

At up to 3750 ppm: Ano-genital distances (absolute and normalized for body) for male or female offspring were unaffected by treatment with test item. A check at PND13 was performed to assess for the presence or absence of nipple/areolae for the male offspring; no nipples were found.

There were no toxicologically relevant macroscopic/pathological findings observed for F1 males or females.

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

Skeletal malformations:

no effects observed

Description (incidence and severity):

There were no toxicologically relevant macroscopic/pathological findings observed for F1 males or females.

Important note:
At 7500 ppm (high dose group), only three F0 females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.

Visceral malformations:

no effects observed

Description (incidence and severity):

Other effects:

no effects observed

Description (incidence and severity):

Details on embryotoxic / teratogenic effects:

A clear effect was noted in females at 7500 ppm. Whereas, in females at 1500 ppm the developmental effects could not be disregarded. No effects were seen at 3750 ppm.

At 7500 ppm: a test item-related severe decrease in gestation index and post-implantation survival index was observed at 7500 ppm. In this high dose group, only three females delivered. Two of them had a total litter loss before first litter check. The third female had only one pup during the period of lactation. As such, no toxicological evaluation for parturition/maternal care, litter size, live birth index, viability index, lactation index, clinical signs, body weights of pups, sex ratio, anogenital distance, areola/nipple retention, clinical biochemistry and macroscopic findings could be performed.
At 1500 ppm: a test item-related decrease in gestation index, post-implantation survival index, live litter size and live birth index was observed. Only 5/10 females produced a healthy normal litter (two females with a total litter loss, two females with a few pups only and one female not pregnant).

It was considered that as test item related developmental effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level and that consequentially, no reproductive/developmental NOAEL could be established. Further investigation was required.

Key result

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Conclusions:

Executive summary:

Toxicity phase results

Reproductive results

Developmental results

Conclusion:

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2021
Report date:: 2021

Materials and methods

Test guidelineopen all close all

Test guideline 1

Qualifier:: according to guideline
Guideline:: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:: no

Test guideline 2

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

Test material

Test material information

Test material form:: liquid
Details on test material:: - Physical state: Liquid
- Storage condition of test material: In refrigerator (2-8°C) protected from light container flushed with nitrogen
- Other: colourless liquid

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) Pre-diet formulation: In refrigerator (2-8°C) protected from light container flushed with nitrogen ; (ii) Formulated-diet: Diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Diet was allowed to thaw before feeding commenced. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Stability under test conditions:
(i) Pre-diet formulation: Stable
(ii) Formulated-diet: Stable (for up to 21 days when stored in a closed container at room temperature and/or when stored frozen at ≤ -15°C in a closed container); formulated-diet was prepared and maintained for 12 days until replaced with diet, that taken from stored diet or freshly prepared diet.
In a preceding dietary formulation and method validation and/or within the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report). Specifically, within the preceding in a dietary formulation and method validation : stability for at least 12 days in an open container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in a closed container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in the freezer (≤ -15°C) in a closed container was demonstrated, over the concentration range 500 to 15000 ppm (full details available in the full study report). Concentration/homogeneity analysis was performed within the definitive study: Duplicate sets of samples (approximately 5 g) were used for concentration/homogeneity analysis, the remaining samples were retained at the testing facility as backup samples. Concentration/homogeneity results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Dietary analyses, during the definitive test confirmed that test item containing diets were prepared accurately and
homogenously.
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.
- 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: No. The test item was directly prepared into formulated diet. By the following:
The test item was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Standard powder rodent diet (certified diet, recognised supplier) was utilized Diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Preliminary purification step (if any): Not applicable.
- Preparation of a nanomaterial dispersion (incl. dilution): 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): Not applicable. Applied in formulated diet.

INFORMATION ON NANOMATERIALS
- Chemical Composition: Not applicable.
- Density: Not applicable.
- Particle size & distribution: Not applicable.
- Specific surface area: Not applicable.
- Isoelectric point: Not applicable.
- Dissolution (rate): Not applicable.

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: Not applicable.
- other information: Not applicable.

Test animals

Species:: rat
Strain:: Wistar
Remarks:: Crl:WI(Han)
Details on species / strain selection:: The species and strain was selected in accordance with the relevant OECD TG 422 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: 287 – 340 g; Females: 196 – 242 g
- Fasting period before study: No.
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in plastic cages (Macrolon MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage housed up to 5 per cage (see above, Macrolon MIV type height 18 cm). Females were individually housed in plastic cages (Macrolon MIII type height 18 cm). During the lactation phase, females were housed in plastic cages (Macrolon MIII type height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Cages contained appropriate environment enrichment and were equipped with water bottles. Group housed males and females and individual housed females, including females during mating, gestation and with litters, were housed in plastic cages containing appropriate bedding and according to relevant legislative requirements. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (dimensions: 48.3 x 26.7 x 20.3 cm) without environmental enrichment, bedding material, food and water.
- Use of restrainers for preventing ingestion (if dermal): Not applicable.
- Diet (e.g. ad libitum): Certified powdered diet, used for treatment (formulated), ad libitum ; during recovery, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: seven days before commencement of treatment. Females: seven days between arrival and start of estrous cycle smears (females) i.e. seven days before start of the pretest period

DETAILS OF FOOD AND WATER QUALITY: Feed: Certified powdered diet, ad libitum – batch numbers and certificates of analysis citations were 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 24°C)
- Humidity (%): 55 ± 15 (or 40 to 70 : actual 48 to 73% ; The values that were outside the targeted range occurred for 8 days with a maximum of 73% and were without a noticeable effect on the clinical condition of the animals, and it was considered without noticeable effect on the outcome of the study).
- Air changes (per hr): > 10 per hour (no recirculation)
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2020-07-29 To: 2020-10-08

Administration / exposure

Route of administration:: oral: feed
Details on route of administration:: The route of administration was in accordance with the relevant OECD TG 422 guideline.
The test item was administered to the appropriate animals by inclusion in the diet ad libitum from Day 1 onwards.
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. 57 days and females that failed to deliver: 42 to 44 days and/or total litter loss: 41 to 42 days.
The amount of test item incorporated in the diet was kept at a constant level in terms of ppm, throughout the pre-mating, mating and post-coitum period. During the lactation period test item concentrations in the diet were adjusted based on historical control data for relative food consumption. After termination, the actual test item intake was estimated based on the body weight and food consumption values.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper.
Vehicle:: unchanged (no vehicle)
Details on oral exposure:: PREPARATION OF DOSING SOLUTIONS:
Not applicable. Dietary study.

DIET PREPARATION
- Rate of preparation of diet (frequency): equivalent to every 12 days ; specifically: diets were prepared freshly for use at room temperature for a maximum of 12 days. Diets were kept at room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use, if not used on the day of preparation. Diet was allowed to thaw before feeding commenced. Any remaining food left after filling the food hoppers was stored at room temperature for a maximum of 12 days for supplementing food during the respective food consumption measurement interval.
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z ; certified diet, recognised supplier)
- Storage temperature of food: room temperature and/or for stored diet (for replacement) : room temperature for a maximum of three weeks in closed bags until use, or stored in the freezer (≤-15°C) for a maximum of three weeks until use. Diet was allowed to thaw before feeding commenced.
- Stability under test conditions:
(i) Pre-diet formulation: Stable
(ii) Formulated-diet: Stable (for up to 21 days when stored in a closed container at room temperature and/or when stored frozen at ≤ -15°C in a closed container); formulated-diet was prepared and maintained for 12 days until replaced with diet, that taken from stored diet or freshly prepared diet.
In a preceding dietary formulation and method validation and/or within the definitive test, the test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report). Specifically, within the preceding in a dietary formulation and method validation : stability for at least 12 days in an open container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in a closed container at room temperature under normal laboratory light conditions was demonstrated. Stability for at least 3 weeks in the freezer (≤ -15°C) in a closed container was demonstrated, over the concentration range 500 to 15000 ppm (full details available in the full study report). Concentration/homogeneity analysis was performed within the definitive study: Duplicate sets of samples (approximately 5 g) were used for concentration/homogeneity analysis, the remaining samples were retained at the testing facility as backup samples. Concentration/homogeneity results were considered acceptable if mean sample concentration results were within or equal to ± 20% for diet of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%. Dietary analyses, during the definitive test confirmed that test item containing diets were prepared accurately and
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not applicable.
- Concentration in vehicle: Not applicable.
- Amount of vehicle (if gavage): Not applicable. Dietary study.
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: - Diet formulations and/or test item administration was examined in a preceding: dietary formulation and method validation and/or within the definitive test (full details available in the full study report).
- The formulated diet analysis consisted of UPLC-TUV analysis with external calibration. The method was calibrated by using calibration standards of the test item response between six nominal concentrations of 20 mg/L and 120 mg/L (working solutions), that were prepared in acetonitrile from two stock solutions (2000 mg/L) within a dedicated dietary formulation analysis report attached to the full study report. These were then subjected to analysis in duplicate by UPLC-TUV. The analytical method was validated (details available within the full study report). For each level, duplicate responses were used. Linear regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficient of correlation (r) was > 0.99.
- Quality Control (QC) samples: Approximately 5 g blank powder diet was spiked with the test item at a target concentration of 1500 or 7500 ppm (using a spiking solution at 4000 mg/L concentration in acetonitrile). After spiking, the test item was allowed to adsorb to the diet for at least 30 minutes. The QC samples were treated similarly as the study samples. mean accuracies of the QC samples were within the criterion range of 80-120%. It demonstrated that the analytical method was adequate for the determination of the test item in the study samples. Actual mean accuracy in target concentrations were : 1500 ppm: 95% and/or 7500 ppm: 98%, respectively.
- Stability: The test item formulated-diet demonstrated adequate stability and homogeneity for up to 21 days when formulations were stored frozen (≤ -15°C) and 12 days when they were stored at ambient room temperature (15 to 25°C). The diet was replaced every 12 days during treatment/exposure (full details available in the full study report).
- Study samples: Samples of approximately 60 g or 250 g were taken from the diets. For determination of accuracy, samples were taken at the random position or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the diets. Diet residue samples were taken from a blank diet which was prepared after routine cleaning of the diet preparation equipment. Duplicate samples of approximately 5 g of the powder diets were accurately weighed into glass vessels. The samples were extracted at 225 rpm with 50 mL acetonitrile. The shaking time was 30 minutes. The solutions were filtered through a 0.2 μm Spartan 30/0.2 RC filter and analysed. If necessary, the samples were further diluted with acetonitrile to obtain concentrations within the calibration range (dilution factors, where applicable reported in the full study report). In the Group 1 (control ; 0 ppm) diets, no test item was detected. The concentrations analysed in the diets of Group 2 (1500 ppm), Group 3 (3750 ppm) and Group 4 (7500 ppm) were in agreement with target concentrations (i.e. mean accuracies between 80% and 120%). Actual: group 2, 3 and 4 accuracies : 92%, 93% and 93%, respectively. The diets of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Actual group 2 and 4 homogeneity CoV : 0.68% and 0.66%, respectively.
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 to 30 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. 57 days and females that failed to deliver: 42 to 44 days and/or total litter loss: 41 to 42 days.
Frequency of treatment:: Continuous via diet
F1 generation were not dosed (were potentially exposed to the test item in utero, via maternal milk, from exposure to maternal urine/feces, or spilled diet from the food hopper).

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 ppm
Remarks:: Control – Group 1; Basal diet

Doses / concentrations 2

Dose / conc.:: 1 500 ppm
Remarks:: Low – Group 2

Doses / concentrations 3

Dose / conc.:: 3 750 ppm
Remarks:: Intermediate – Group 3

Doses / concentrations 4

Dose / conc.:: 7 500 ppm
Remarks:: High – Group 4

Doses / concentrations 5

Dose / conc.:: 99 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for males for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Doses / concentrations 6

Dose / conc.:: 250 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for males for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Doses / concentrations 7

Dose / conc.:: 501 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for males for 7500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Doses / concentrations 8

Dose / conc.:: 109 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for females for 1500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Doses / concentrations 9

Dose / conc.:: 275 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for females for 3750 ppm group
(mean of means of all periods, weighed for number of measurement days per period)

Doses / concentrations 10

Dose / conc.:: 534 mg/kg bw/day (actual dose received)
Remarks:: Overall Mean for females for 7500 ppm group
(mean of means of all periods, weighed for number of measurement days per period)
note: data of only one female was present during the lactation period (mean intake 281 mg/kg bw/day) and therefore this period was not included in the Mean of means calculation.

No. of animals per sex per dose:: 10 per sex per dose (10 male / 10 female) with satelliete 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/feces, or spilled diet from the food hopper).

Males:
Control (toxicity test) = 10
Control (recovery phase) = 5
1500 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5

Females:
Control (toxicity test) = 10
Control (recovery period) = 5
1500 ppm (toxicity test) = 10
3750 ppm (toxicity test) = 10
7500 ppm (toxicity test) = 5
7500 ppm (recovery period) = 5
Control animals:: yes, plain diet
Details on study design:: - Dose selection rationale: The dose levels selected for investigation in this study (0, 1500, 3750 and 7500 ppm) by dietary administration were chosen based upon the results obtained in a 14-day dose range finding study (full details available in the full study report). Samples for diet analysis were not collected during the dose range finder, as concentration, homogeneity, and stability analysis was not performed. However, to limit the impact, the diet preparation was performed with approved procedures and documented in detail. Preparations were visually inspected for homogeneity prior to use and all preparations were used within 12 days after preparation of the diets. Homogeneity and stability of the diet under test conditions was demonstrated in the analytical method development and validation study (cited in the full study report ; also used within the definitive study where concentration analysis was performed). Dose levels in the 14-day DRF test were: Group 1: 7500 ppm and Group 2: 15000 ppm ; administered by inclusion in the diet ad libitum from Day 1 onwards for a minimum of 10 days. Based upon the results of the acute oral toxicity test (anon., 2020) and/or considering the structural class of the substance, staggered start approach was chosen for this dose range finder study for ethical reasons. The study was initiated with Group 1. At 7500 ppm no clinical signs were observed. Food consumption during Week 1 of administration (especially Days 1-4) was very low, causing a significant body weight loss in 3/3 females. During Week 2 of administration food consumption completely recovered to normal levels, and body weights recovered to levels just below the historical control (HCD) range. Based on this data, 15000 ppm was selected as dose level for Group 2. At 7500 ppm: there was no mortality, no significant clinical findings were apparent. There was severe body weight loss over Days 1-4 of treatment, which recovered to start weight on Day 8-11. Body weight remained slightly low at the end of treatment. Food consumption was severely low on Days 1-4, followed by recovery to normal values from Days 8-11 onwards. There was no macroscopic abnormalities noted. Liver and kidney weights were considered to be normal. The mean test item intake was considered to be 490 mg/kg bw/day (at 7500 ppm nominal). At 15000 ppm: 3/3 females were sacrificed in extremis on Day 11. Within clinical signs, there was hunched posture and piloerection starting at Day 6-7 in 3/3 females. There was severe body weight loss from Day 4 of treatment onwards and/or food consumption was slightly low over Days 1-4 (actual value lower due to food spillage); severely low from Day 4-8, followed by partial recovery from Days 8-11. There was no macroscopic abnormalities noted. Organ weights (kidney and liver weights) were stated as ‘not available’ (not determined since terminated at in extremis not at ‘scheduled necropsy’). The mean test item intake was considered to be 810 mg/kg bw/day (at 15000 ppm nominal).
- 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. Deviation: Recovery Males were not fasted before clinical pathology blood collection at the end of treatment. It was considered within the study: Clinical Pathology values were generally within the same range as for Main males (except for glucose levels). As concurrent control Recovery Males were also not fasted, sufficient evaluation could be performed. It was considered this was not a significant deviation.
- 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

Examinations

Observations and examinations performed and frequency:: 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: Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery periods up to the day prior to necropsy. 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: Yes. Individual Food Consumption (g/animal/day), Relative Food Consumption (g/Kg body weight/day) and/or test item intake (mg substance /Kg body weight/day) were all determined. Mean daily consumption per animal (g/animal/day) was calculated for each phase,
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes.
- 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.

FOOD EFFICIENCY: Yes.
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No. - Body weight gain % was determined. Calculated against the body weight on Day 1 (pre-mating and lactation periods) or Day 0 (post-coitum period).

WATER CONSUMPTION: Yes
- Time schedule for examinations: 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.

OPHTHALMOSCOPIC EXAMINATION: No.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy for all test and control group individuals (or when humanely terminated in extremis).
- Anaesthetic used for blood collection: Yes. isoflurane (recognised supplier)
- Animals fasted: Yes. Overnight (maximum 24 hours). Females were not fasted.
- How many animals: All animals. See above for fasting.
- Parameters checked: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices – including: mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), Total leukocyte count (WBC), Differential leukocyte count – including: neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas), Platelet count (PLT), Reticulocyte count (Retic). Additionally: Prothrombin time (PT) was assessed and Activated partial thromboplastin time (APTT) was assessed.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day of scheduled necropsy for all test and control group individuals (or when humanely terminate in extremis).
- Animals fasted: Yes. Overnight (maximum 24 hours). All males (Main and Recovery) were fasted. Main Females were not fasted. Recovery females were fasted.
- How many animals: All animals, as applicable. See above for fasting.
- Parameters checked: Urea, Aspartate aminotransferase (ASAT), Glucose, Alanine aminotransferase (ALAT), Total protein (Tot.Prot.), Alkaline phosphatase (AP), Albumin, Creatinine (Creat), Total cholesterol (Chol), Sodium (Na+), Total bilirubin (Bili), Potassium (K+), Chloride (Cl-), Bile acids, Calcium (Ca++), Inorganic phosphate (P)

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes. Was conducted as part of ‘special evaluations’
- Time schedule for examinations: Functional performance tests were also performed on selected animals during Week 4, together with an assessment of sensory reactivity to different stimuli. This involved the selection of 5 males during Week 4, and all Recovery females (Groups 1 to 4) then the selected 5 Main females (Groups 1 to 3) during the last week of lactation (i.e. PND 6-13). Group 4 Main females were tested days 24-26 post-coitum. These tests were performed after dosing, after completion of clinical observations and included: hearing ability, pupillary reflex, static righting reflex, fore and hind limb strength, locomotor activity.
- Dose groups that were examined: All.
- Battery of functions tested: sensory activity / grip strength / motor activity – see above for further information.

IMMUNOLOGY: No

OTHER: Additional post-termination observations were made at necropsy.

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 (Main and Recovery) 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. Since suspected treatment related effects were obtained in the first 14 days of treatment, daily vaginal lavage was performed for Recovery females during the treatment-free recovery period to investigate reversibility of effect.
- 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 terminated in extremis).

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).
Sacrifice and pathology:: GROSS PATHOLOGY: Yes. All Selected Main Animals and all Recovery Animals.
- 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: Yes. All Selected Main Animals and all Recovery Animals.
- 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.
Statistics:: Refer to 'Any other information on materials and methods incl. tables' for detailed information on statistics.

Results and discussion

Results of examinations

Clinical signs:: no effects observed
Description (incidence and severity):: There were no significant clinical signs observed considered that were related to treatment during treatment, gestation or lactation periods.

Any clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain, housed and treated under the conditions in the study. There were no apparent dose-related trends and at the incidence observed, these were considered to be unrelated to treatment with the test item.

For the purposes of reporting : For males, “Repro period” represented the mating phase. For females, “Repro period” represented the mating, post-coitum and lactation phase.
Mortality:: no mortality observed
Description (incidence):: There were no treatment related mortalities.

Two females of the 1500 ppm group (female #70 and #72) and two females of the 7500 ppm group (female #86 and #87) were euthanized on Lactation Day 1, as they had a total litter loss.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: At 7500 ppm: treatment-related changes in body weight and body weight gain were observed in males and females were observed.

In males, body weight stasis was observed in the first week of treatment, followed by a lower body weight gain when compared to concurrent controls in the following 3 weeks of treatment. At the end of the treatment period, mean body weight was x0.88 of control. In the subsequent treatment-free period of 14 days, mean body weight recovered to x0.93 of control. Considering the magnitude of the changes, these findings were considered adverse.

In Main females lower body weight gain was noted when compared to concurrent controls from Day 4 post-coitum onwards, resulting in a mean body weight of x0.81 of control at the end of the gestation period. Possible test item-related effects on body weight during lactation could not be assessed since only one lactating female with a single pup was available in the 7500 ppm group. In Recovery females, body weight gain was decreased on Days 15 and 22 of the repro period, resulting in decreased body weights (up to x0.93 of control on Day 15 of the repro period). At the end of the treatment period and the 14-days treatment-free recovery period, mean body weight was respectively 96% and 97% of control. As body weights of Recovery females recovered and considering the magnitude of change, these were not considered toxicologically relevant.

At up to 3750 ppm: body weight and body weight gain were considered unaffected by treatment in males and females.

At 3750 ppm: in males, mean body weight gain was decreased on Days 1 and 8 of the mating period, which resulted in slightly decreased absolute body weights (up to x0.95 of control on Day 8 of the mating period, not statistically significant). In view of the slight nature of this change, it was regarded not to be toxicologically relevant. At 1500 ppm and 3750 ppm, in females, decreased body weight gain was recorded on respectively Day 20 and Day 17 post-coitum, resulting in slightly decreased mean body weights at the end of gestation (x0.93 and x0.94 of control, respectively). In absence of a dose-related trend, no toxicological relevance was attached to this finding. No treatment-related changes were recorded during the lactation period.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: At 7500 ppm: in both males and females, food consumption was slightly decreased during the first week of treatment (not statistically significant) which subsequently recovered.

In males, a trend towards decreased absolute food consumption was recorded throughout the treatment period (up to x0.84 of control during the first week of mating). In females, decreased absolute and relative food consumption was recorded on several days during the post-coitum period (up to x0.78 of control for absolute food consumption). This was considered treatment-related. The lower food consumption in females during the first week of treatment and over post-coitum Days 0-4 was transient and considered likely to be palatability-related and therefore, no toxicological relevance was attached to it.

Note that data of only one female was present during the lactation period and therefore food consumption could not be assessed.

In Recovery males and (nulliparous) Recovery females, food consumption before or after correction for body weight was similar to the control level over the repro period and in the treatment-free recovery period thereafter.
Food efficiency:: no effects observed
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:: no effects observed
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 male (#35) at 3750 ppm and/or one female #52 in the control group, after treatment and/or one male #13 in the recovery control group, only post termination.
Haematological findings:: effects observed, non-treatment-related
Description (incidence and severity):: Assessment of haematological parameters at the end of the treatment period revealed no treatment related effects (within assessable groups).

At 7500 ppm: except for one lactating female (#90), no matching controls were available for Main females due to their different physiological status (two with total litter loss, two with implantation sites only) and shorter (about one week) treatment period. Therefore no summary tables for Group 4 Main females were available and toxicological evaluation at 7500 ppm was performed for (nulliparous) Recovery females only.

No toxicologically relevant changes were noted in haematological parameters in males up to 7500 ppm, (lactating) Main females up to 3750 ppm and (nulliparous) Recovery females at 7500 ppm.

Statistically significant changes in white blood cell (WBC), lymphocyte and large unstained cell (LUC) counts in Main male rats at 1500 and/or 3750 ppm were considered to be unrelated to treatment with the test item as these occurred in the absence of a dose-related trend.

Statistically significant changes in WBC, lymphocyte, monocyte and LUC counts in Recovery male rats and red blood cell (RBC) counts in Recovery female rats were considered to be unrelated to treatment with the test item as these occurred at the end of the recovery period only.

At up to 7500 ppm, for coagulation parameters: treated male rats and (nulliparous) Recovery females and/or (lactating) Main females up to 3750 ppm were considered not to have been affected by treatment. See notes above, (lactating) Main females at 7500 ppm could not be assessed due to absence of equivalent controls.

At 7500 ppm, statistically significantly longer prothrombin time (PT) in males at 7500 ppm was considered the result of a slightly low control mean and therefore unrelated to treatment with the test item. [Historic control data: F0-males (period 2017-2019): PT (s): mean = 18.2; P5 – P95 = 16.30-20.70 (n=321)].
Clinical biochemistry findings:: effects observed, treatment-related
Description (incidence and severity):: Statistically significant clinical chemistry changes distinguished treated from control animals at the end of the treatment period. No changes were recorded after the 14-day treatment-free period. There was evidence of recovery.

At 7500 ppm: except for one lactating female (#90), no matching controls were available for Main females due to their different physiological status (two with total litter loss, two with implantation sites only) and shorter (about one week) treatment period. Therefore no summary tables for Group 4 Main females were available and toxicological evaluation at 7500 ppm was performed for (nulliparous) Recovery females only.

At up to 7500 ppm: in males, decreased total bilirubin concentrations starting at 1500 ppm and up to x0.61 of control at 7500 ppm, was observed. Within Recovery females at 7500 ppm they were x0.68x of control at the end of treatment. In males at 1500 and 3750 ppm this was considered the result of slightly high control values. [Historic Control Data: F0-males (period 2017-2019): Total bilirubin (umol/L): mean = 2.3; P5 – P95 = 1.60-3.10 (n=355)]. In males and females at 7500 ppm this change was regarded treatment-related.

At 7500 ppm: in males, Increased creatinine concentration x1.21x of control was observed, which was considered treatment-related. The decreased cholesterol concentration in males x0.76x of control was considered not toxicologically relevant based on the direction of this change.

Decreased potassium concentrations in females at 1500 and 3750 ppm were considered not toxicologically relevant, as the result of a slightly high control mean. [Historic Control Data: F0-females (period 2017-2019): Potassium (mmol/L): mean = 4.54; P5 – P95 = 3.410-5.320 (n=325)].
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. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

At 3750 ppm, in females, increased total movements and ambulations were recorded (x1.33 and x1.44 of control, respectively). As mean values remained within the historical control range no toxicological relevance was attached to this finding. Note that no matching controls were available for Group 4 females due to their different physiological state (i.e. failed to deliver) and shorter (about one week) treatment period. Motor activity in Group 4 and (nulliparous) Recovery females was considered within the normal range for rats of this age and strain.
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: At 7500 ppm: In males, potential test item related lower thymus weights (absolute and relative to body weights) and higher liver weights (relative to body weight were noted). The liver weight findings achieved statistical significance. Furthermore some organ weight differences were statistically significant when compared to the control group weight: absolute testes and prostate gland weights and relative brain weight. However, they were considered to be the result of a test item-related effect on final body weight (0.86x of control). There was no test item-related effect on the final body weight and organ weights of the Recovery males.

In females, assessment could not be completed. Selected females of the 7500 ppm group had a shorter treatment duration and different physiological status (implantation sites only or total litter loss), compared to the selected females of the control group (except for Female #90). Therefore, no comparison of organ weight of the 7500 ppm Main females with the control group could be made and no toxicological evaluation could be performed.

At up to 3750 ppm: there were not test item related effects on organ weights of main female.

Any other organ weight differences, including those that reached statistical significance (relative to body weight of the liver of 7500 ppm Recovery females) were considered not to be test item-related due to the direction of the change, lack of dose-related pattern, lack of microscopic correlate and/or general overlap and variability in individual values.

For further information see table 2. and/or ‘histopathological findings’. It was considered the findings of the bone marrow and thymus were most likely related to a test item-related effect on the final body weight. In Main males at 7500 ppm, the possible higher liver weight was without microscopic correlate. Therefore, the findings of the bone marrow, thymus and liver, were considered non-adverse and showed full recovery after a 14-day treatment-free period (Kerlin et al., 2016; Palazzi et al., 2016).
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: At 7500 ppm: a test item-related macroscopic finding was noted in a single female of the Main group, consisting of an enlarged spleen. The microscopic correlate was extramedullary haematopoiesis (marked degree).

For further information see table 4. and/or ‘histopathological findings’.

The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: At 7500 ppm: In males, test item-related microscopic findings after treatment were noted in the epididymides, testes, thymus and bone marrow. Degeneration of germ cells of tubules (mainly round spermatids) and spermatid retention was noted at 7500 ppm in the testes of a single Main male (#39), both findings slight, as well as moderate luminal cellular debris of the epididymides. Decreased lymphoid cellularity in the thymus was noted at a minimal degree in Main males. An increased incidence and severity of increased adipocytes in the bone marrow (sternum and femur) was noted in Main males up to slight degree. All of the listed test item-related findings of 7500 ppm Main males were absent in 7500 ppm Recovery males. For further information see table 3.

In females, test item-related microscopic findings after treatment were noted in the liver, spleen, kidneys, bone and bone marrow of the Main females and/or Recovery females. An increased incidence and severity of extramedullary haematopoiesis of the spleen was noted in Main females. In the female with the highest severity (#88, marked degree) extramedullary haematopoiesis was also noted in the liver at a slight degree. The extramedullary haematopoiesis in the Recovery females (minimal degree) was within background range values. Moderate degeneration and anatomically related slight dilation of tubules was noted in the kidneys of a single 7500 ppm Main female (#86). The degeneration was characterized by basophilia and sloughing of necrotic cells into the tubular lumen. Increased adipocytes (sternum and/or femur) and/or increased hematopoietic cellularity was noted in the bone marrow of Main females and was absent in Recovery females. Increased bone (femur, up to slight degree) was noted in both Main females and in Recovery females. There was an increase in trabecular bone, particularly below the growth plate at the metaphyseal side. For further information see table 4.

Possible test item-related microscopic findings were present in the female reproductive organs and mammary gland of 7500 ppm Main females and are described in the reproductive performance/reproduction sections. Findings of note: Abnormal estrous cycles (including inactive uterus epithelium and/or increased mucification of the vaginal epithelium) were noted in one Recovery female of the control group (#61) and in one Recovery female (#100). This was considered to be related to the frequent lavage procedure used in the Recovery females. For further information see table 5. and/or relevant reproduction/developmental toxicity sections.

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Overall, it was considered that:
At 7500 ppm, increased bone (femur) was noted in Main and Recovery females. This finding alone, was at the recorded minimal or slight degree considered as non-adverse. Relation to the abnormal estrus cycle and/or poor reproductive performance noted in the current study could not be established. The bone marrow (sternum and/or femur) of Main males and females at showed a subtle increase in incidence and severity of increased adipocytes (up slight degree). A decreased lymphoid cellularity (minimal) of the thymus, correlating with a lower organ weight, was noted in Main males. These findings of the bone marrow and thymus were most likely related to a test item-related effect on the final body weight. Finally, a possible higher liver weight, without microscopic correlate, was observed in Main males. The findings of the bone marrow, thymus and liver, were considered non-adverse and showed full recovery after a 14-day treatment-free period (Kerlin et al., 2016; Palazzi et al., 2016).

For Main females at 7500 ppm, no matching controls were available for due to their different physiological status and shorter treatment period, and therefore no toxicological evaluation could not be performed.

No toxicologically significant effects were observed at up to 3750 ppm, however it was considered that within reproductive parameters, that further investigation was required.

Note: the reproduction and developmental NOAEL could not be established in the OECD TG 422 study, as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation. The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test (OECD TG 421) and were determined to be at least 3750 ppm.
Histopathological findings: neoplastic:: no effects observed
Other effects:: effects observed, treatment-related
Description (incidence and severity):: 1. Thyroid Hormone Assessment:
(i) F0 generation :
1. Serum levels of T4 in F0-males were dose dependently lower by x0.60 and x0.19 of control (achieving statistical significance) at 3750 ppm and 7500 ppm, respectively. In Recovery males, no difference was recorded after the 14-day recovery period. There was evidence of recovery.
2. Serum levels of T4 in F0-females, at 7500 ppm were lowered at x0.55 of control (achieving statistical significance). In Recovery females at the end of treatment they were x0.23 of control (achieving statistical significance), which was considered treatment related. In Recovery females, increased T4 serum levels were recorded x1.52 of control (achieving statistical significance), after the end of the 14-day recovery period.
3. TSH levels were decreased in F0-males at all dose levels x0.81, x0.70 and x0.81 of control in Groups 2, 3 and 4 (or 1500, 3750 and 7500 ppm, respectively). At 7500 ppm, in nulliparous Recovery females they were x0.72 of control, while at 3750 ppm increased TSH in Main F0-females was recorded x1.83 of control. After a 14-day treatment-free period TSH levels were increased in Recovery males at x1.72 of control, while decreased in Recovery females x0.77 of control.

Within the study there was no correlates to the Thyroid in gross pathology, organ weights or histopathology. Consequently, T3 was not further analysed.

In this OECD TG 422 study, marked changes of total T4 and TSH were observed in low dose groups (males only) and in mid and high dose groups (both sexes). However, under the conditions of this screening study the toxicological relevance is not clear, and no adverse effect or other correlates were observed that could be linked to these changes. Therefore these were not taken into account when determining the parental NOAEL.

Since the reproduction and developmental NOAEL could not be established within this study, as test item related effects were observed at the highest dose level, with no significant effects in the mid dose group and unclear effects at the lowest dose level. This required further investigation. The reproductive/developmental NOAELs were clarified and established in subsequent reproduction/developmental toxicity screening test (OECD TG 421) and were determined to be at least 3750 ppm. T4 and/or TSH were further investigated therein.

(ii) F1 offspring:
Serum T4 levels in PND 14-16 males and females were considered to be unaffected by treatment.

At 1500 and 3750 ppm, slightly increased serum T4 levels in female pups up to x1.16 of control were considered not related to treatment with the test item considering the magnitude
of the change (≤ 20%).

In the related gross pathology/histopathology:

The thyroids of the F1 offspring were considered unaffected by treatment up to 3750 ppm.

There were no other macroscopic findings finding observed in the offspring that were attributable to parental treatment with the test item up to 3750 ppm.
Details on results:: In relation to Thyroid Hormone Assessment - see cross-referenced study OECD TG 421 (2021). Further comments are provided therein as part of applicant assessment with relevant literature citations.

Reference: 'Toxicity to reproduction - OECD 421 - 2021' - section: F0 and/or F1 'other effects' : Thyroid Hormone Assessment

Effect levels

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1

: Key result
Dose descriptor:: NOAEL
Effect level:: >= 3 750 ppm
Based on:: test mat.
Sex:: male/female
Basis for effect level:: body weight and weight gain; clinical biochemistry

2

: Key result
Dose descriptor:: NOAEL
Effect level:: >= 250 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: male
Basis for effect level:: body weight and weight gain; clinical biochemistry

3

: Key result
Dose descriptor:: NOAEL
Effect level:: >= 275 mg/kg bw/day (actual dose received)
Based on:: test mat.
Sex:: female
Basis for effect level:: body weight and weight gain; clinical biochemistry

Target system / organ toxicity

: Key result
Critical effects observed:: no

Any other information on results incl. tables

Table 1.0 : Test Item Intake within the Dietary Study

		Mean over means intake [mg test item/kg body weight] (mean range indicated in bracktes)
	Group Number	2		3		4
	Nominal dietary inclusion level (ppm)	1500		3750		7500
Sex	Study Period
Male	Pre-mating	103	(102-105)	261	(258-264)	512	(473-551)
	Post-mating	95	(93-98)	238	(227-250)	489	(481-497)
	Mean of means #a	99		250		501
Female	Pre-mating	109	(108-110)	261	(254-268)	555	(459-650)
	Post-coitum	111	(102-121)	278	(263-289)	519	(452-565)
	Repro period (Recovery females)	n.a.		n.a.		480	(447-516)
	Lactation	104	(95-112)	211	(274-296)	281 #b	(246-317)
	Mean of means #a	109		275		534

where:

#a : Mean of means of all periods, weighed for number of measurement days per period:

Males: ((14 x mean premating) + (14 x mean mating)) / 28

Females: ((14 x mean premating) + (20 x mean post-coitum) + (12 x mean lactation)) / 46

#b : Data of only one female was present during the lactation period and therefore this period was not included in the Mean of means calculation.

For Main males, “Repro period” represents the mating phase.

For Main females, “Repro period” represents the mating, post-coitum and lactation phase.

For Recovery animals, the “Premating period” and “Repro period” represents the treatment phase.

Notes:

Test item intake was calculated as concentration of test item in diet (ppm) against relative food consumption.

Table 2.0 : Mean Percent Thymus and Liver Weight Differences from Control Groups

		Main males			Recovery males
Dose level (ppm)		1500	3750	7500	7500
THYMUS	Absolute	-16	9	-36	-1
	Relative to B.W.	-9	17	-27	6
LIVER	Absolute	-3	-4	-3	-9
	Relative to B.W.	4	3	12 **	-3

Where:

B.W. : body weight

** : P < 0.01

Table 3.0 : Summary Test Item-Related Microscopic Findings – Scheduled Termination Males.

	Main males				Recovery males
Dose level (ppm)	0	1500	3750	7500	0	7500
EPIDIDYMIDES #a	6	5	6	5	5	5
Cell debris
Moderate	-	-	-	1	-	-

TESTES #a	6	5	6	5	5	5
Spermatid retention
Slight	-	-	-	1	-	-
Degeneration tubule
Slight	-	-	-	1	-	-

THYMUS #a	5	5	5	5	5	5
Decreased cellularity
Minimal	-	-	-	2	-	-

BONE MARROW, STERNUM #a	5	5	5	5	4	5
Increased adipocytes
Minimal	-	-	-	1	-	-
Slight	-	-	-	1	-	-

BONE MARROW, FEMUR #a	5	5	5	5	5	5
Increased adipocytes
Minimal	1	-	1	1	1	-
Slight	-	-	-	1	-	-

#a : number of tissues examined from each group

Table 4.0 : Summary Test Item-Related Microscopic Findings – Scheduled Termination Females.

	Main females				Recovery females
Dose level (ppm)	0	1500	3750	7500	0	7500
LIVER #a	5	5	5	5	5	5
Extramedullary haematopoiesis
Slight	-	-	-	1	-	-

SPLEEN #a	5	5	5	5	5	5
Extramedullary haematopoiesis
Minimal	3	2	5	1	2	3
Slight	1	-	-	1	-	-
Moderate	-	-	-	1	-	-
Marked	-	-	-	1	-	-

KIDNEYS #a	5	5	5	5	5	5
Degeneration tubule
Moderate	-	-	-	1	-	-
Dilation tubule
Slight	-	-	-	1	-	-

BONE MARROW, STERNUM #a	5	5	5	5	5	5
Increased adipocytes
Minimal	-	-	-	3	1	-
Slight	-	-	-	1	-	-
Increased cellularity
Minimal	-	-	-	1	-	-
Slight	-	-	-	1	-	-

BONE MARROW, FEMUR #a	5	5	5	5	5	5
Increased adipocytes
Minimal	-	-	-	1	1	-
Slight	-	-	-	-	-	-
Increased cellularity
Minimal	-	-	-	-	-	-
Slight	-	-	-	1	-	-

BONE, FEMUR #a	5	5	5	5	5	5
Increased bone
Minimal	-	-	-	3	-	3
Slight	-	-	-	2	-	1

#a : number of tissues examined from each group

Table 5.0 : Summary of In-Life Reason for Males that Failed to Sire and Females that Failed to Deliver Healthy Pups.

Group	Dose level (ppm)	Female/Male number	In-life Reason
1	0	58 / 08	Not pregnant
2	1500	68 / 18 70 / - 72 / -	Not pregnant Total litter loss Total litter loss
3	3750	76 / 26 82 / 32	Not pregnant Not pregnant
4	7500	86 / - 87 / - 88 / - 89 / - 91 / - 92 / - 93 / - 94 / - 95 / -	Total litter loss Total litter loss Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only Implantation sites only

Note: For further information relevant reproduction/developmental toxicity sections.

Applicant's summary and conclusion

Conclusions:

Executive summary:

Toxicity phase results

Reproductive results

Developmental results

Conclusion:

Refer to ‘Toxicity to reproduction’ section for further information.

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2-methylpentyl 2-hydroxybenzoate

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