5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males were 10-12 weeks old and females were 12-14 weeks old.
- Weight at study initiation: males weighed between 246 and 312 g and females weighed between 204 and 246 g.
- Housing: On arrival and following the pretest (females only) and pre-mating period, Main 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). Recovery males and females were housed as such during the entire study period. During the mating phase, Main males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Main Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, Main females were housed in Macrolon plastic cages (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 be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water. The cages containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals had no access to food for a maximum of 2 hours.
- Water: Municipal tap water was freely available to each animal via water bottles.
- Acclimation period: 5 days prior to start of the pretest period (females) or 5 days before the commencement of dosing (males)

DETAILS OF FOOD AND WATER QUALITY:
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 22
- Humidity (%): 45-71
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily, were protected from light and dosed within 5 hours after adding the vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Trial preparations were performed at the Test Facility prior to conduct of the Dose Range Finding Study to select the suitable vehicle and to establish a suitable formulation procedure. Ultimately, the vehicle (corn oil) was selected by the Sponsor, i.e. the same vehicle as used in the Dose Range Finding Study.
- Concentration in vehicle: 25, 75, 250 mg/kg
- Amount of vehicle (if gavage): 4 ml/kg

Details on mating procedure:

After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
A maximum of 14 days was allowed for mating, after which females who did not show evidence of mating were separated from their males.
For two couples (male/female nos. 13/67 and 21/71), mating was not confirmed in first instance. As sperm cells were detected in the vaginal lavage during re-evaluation of the lavages, these couples were separated 9 and 12 days after the actual mating date. The actual mating date was designated Day 0 post-coitum.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the Group 1 formulation (control group), no test item was detected. The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Duration of treatment / exposure:

The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Main males and Recovery males were treated for 29 days, up to and including the day before scheduled necropsy. This included a minimum of two weeks prior to mating and during the mating period. For both Main and Recovery males treatment ended one day before scheduled necropsy of Main males. Main females that delivered were treated for 50-56 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 before scheduled necropsy. Recovery females were treated during the same period as Main females, until at least the first scheduled necropsy of Main females. Females which failed to deliver or had a total litter loss were treated for 42 days.

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 plus additional 5 in control and high dose for recovery investigations

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results of a 14-day oral range finding study in the rat. The high dose level was the limit dose according to the guidelines. The other dose levels were chosen to provide an appropriate spacing.
- Post-exposure recovery period in satellite groups: 14 days

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing and recovery period. During the dosing period, these observations were performed after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finder). The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

BODY WEIGHT: Yes
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. 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 fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured weekly, except for Main males and Main females which were housed together for mating and for Main females without evidence of mating. Food consumption of mated Main 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: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5/sex of main animals and all recovery animals
- Parameters examined: White blood cells (WBC), Neutrophil (absolute), Lymphocyte (absolute), Monocyte (absolute), Eosinophil (absolute), Basophil (absolute), Red blood cells, Reticulocyte (absolute), Red Blood Cell Distribution Width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 5/sex of main animals and all recovery animals
- Parameters examined: Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Gamma glutamyl transpeptidase (GGT), Total protein, Albumin, Total Bilirubin, Bile Acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate (Inorg. Phos),

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional tests were performed on the selected 5 Main males and all Recovery males during Week 4 of treatment and the selected 5 Main females during the last week of lactation (i.e. on PND 9, 11 or 12), and all Recovery females were tested on the first day a Main female was tested. These tests were performed after dosing, after completion of clinical observations (including arena observation, if applicable). The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

IMMUNOLOGY: No

THOYROID HORMONE MEASUREMENTS: Yes
Blood samples were processed for serum, and serum was analyzed for Thyroxine (T4). Measurement of T4 was conducted for F0-males and PND 13-15 pups. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no treatment-related changes in T4 were noted in F0-males, no adverse effects on thyroid histopathology and no treatment related changes in thyroid weight were recorded. Assessment of T4 for PND 4 pups and TSH for PND 13-15 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 13-15.

Oestrous cyclicity (parental animals):

Daily vaginal lavage was performed for all females (Main and Recovery) during 14 days prior to treatment (pretest period) and the first 14 days of treatment. For Main females, daily vaginal lavage was continued during mating until evidence of copulation was observed. On the day of necropsy, a vaginal lavage was also taken from Main and Recovery females to determine the stage of estrus. This was done for all females.

Sperm parameters (parental animals):

For testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage- specificity of testicular findings were noted.

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 prevents 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:
Mortality/Moribundity, Clinical Observations, number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies ,body weights, anogenital distance, nipple retention

SAMPLE COLLECTION
Blood of F1-animals was collected on PND 4 and PND 13-15, if possible. On PND 4 at culling, blood was collected from two surplus pups per litter (if possible) by decapitation, between 7.00 and 10.30 a.m., and samples were pooled. If available, blood was collected from one male and one female pup. If only one surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
On PND 13-15, blood was collected from two pups per litter (from one male and one female). Blood was drawn, between 7.00 and 10.30 a.m., by aorta puncture under anaesthesia using isoflurane as part of the necropsy procedure. Measurement of T4 was conducted for PND 13-15 pups. Assessment of T4 for PND 4 pups and TSH for PND 13-15 pups was considered not relevant because no treatment-related changes in T4 were noted in pups at PND 13-15.

GROSS EXAMINATION OF DEAD PUPS:
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.
On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation. From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 13-15. 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. In addition, blood was collected from two pups per litter, and the thyroid from two pups per litter (if possible one male and one female pup) were preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.

Postmortem examinations (parental animals):

GROSS PATHOLOGY
All animals (both Main and Recovery) were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. Scheduled necropsies were conducted on the following days:
- Main Males (which sired or failed to sire): Following completion of the mating period (a minimum of 28 days of administration).
- Recovery males: After the recovery period of at least 14 days, which is at least 14 days after the scheduled necropsy of Main males.
- Main Females which delivered: PND 14-16.
- Main Females which failed to deliver but showed evidence of mating (Nos. 84, 91 and 92): Post-coitum Days 27.
- Recovery females: After the recovery period of at least 14 days, which is at least 14 days after the first scheduled necropsy of Main females.
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.

ORGAN WEIGHTS
The organs identified in the table below were weighed at necropsy for all scheduled euthanasia animals. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of each organ of a pair was determined. Organ to body weight ratio (using the terminal body weight) were calculated.

HISTOLOGY
Representative samples of the tissues identified in table 3 below were collected from all animals and preserved in 10% neutral buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands), unless otherwise indicated. The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
- Selected Parental animals and all Recovery animals: Tissues identified in Table 3 (except animal identification, aorta, nasopharynx, esophagus, harderian gland, lacrimal gland, salivary gland, female mammary gland, larynx, optic nerve, pancreas, skin and tongue.
- Males that failed to sire (except for males which were selected) and females that failed to delivery pups: Cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina.
- Remaining animals: Gross lesions/masses.

HISTOPATHOLOGY
Reproductive organs (cervix, coagulation gland, epididymides, ovaries, prostate gland, seminal vesicles, testes, uterus, and vagina):
- All Main animals of the control and high dose group.
- All males that failed to sire to examine staging of spermatogenesis and histopathology of interstitial cell structure.
- All females that failed to deliver pups.
For ovaries, a qualitative evaluation of 1 section from each ovary was made.
For testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage- specificity of testicular findings were noted.
Remaining organs:
- First five males and lactating females of the control and high dose group.
- Gross lesions for all Main and Recovery animals.
Histopathological evaluation was performed by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 2 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Reproductive indices:

Mating, Precoital Time, ferility index, gestation index, duration of gestation

Offspring viability indices:

Post-implantation survival index, Live birth index, Percentage live males at First Litter Check, Percentage live females at First Litter Check, Viability index, Lactation index

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Salivation seen after dosing among animals of all dose groups, including controls, was not considered toxicologically relevant, taking into account the nature and minor severity of the effect, the absence of a dose-relationship and its time of occurrence (i.e. after dosing). Incidental findings that were noted occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were not considered to be signs of toxicological relevance.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight gain were not considered to have been affected by treatment. The statistically significantly higher body weight and/or body weight gain of non-mated recovery females at 1000 mg/kg bw/day on a few days during the treatment period were considered to be unrelated to treatment, as the differences were small and since body weight of high dose pregnant females was not affected. This variation was ascribed to the lower number of females after Day 1 of the Repro period (n=5) compared to Day 1 (n=15), whereby the slightly higher mean body weight of Group 4 recovery females compared to controls was considered to have occurred by chance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption before or after correction for body weight was not considered to have been affected by treatment. Any statistically significant changes in food consumption before or after correction for body weight were considered to be unrelated to treatment since no trend was apparent regarding dose and/or duration of treatment.

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematological parameters of treated rats were considered not to have been affected by treatment. Any statistically significant alterations in hematological parameters (higher reticulocyte counts in males of the recovery period, lower erythrocyte counts in main group females at 100 mg/kg bw/day, lower neutrophil counts and MCHC values in females at the end of the recovery period) were not considered to be related to administration of the test item due to the minimal magnitude of the change, absence of a dose response, and/or absence of similar variations at the end of treatment. Coagulation parameters of treated rats were not considered to have been affected by treatment. The statistically significant higher prothrombin time (PT) in Recovery females at 1000 mg/kg bw/day at the end of treatment was not considered to be related to treatment as this finding was not noted in males and Main females at the end of treatment and since the mean remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical biochemistry parameters of treated rats were not considered to have been affected by treatment. The statistically significantly higher urea values for Main group males at 1000 mg/kg bw/day at the end of treatment (and a similar but statistically non-significant change in this parameter in males at 300 mg/kg bw/day) was not considered to be related to treatment. Means remained within the range considered normal for rats of this age and strain and control mean was considered to be slightly low based on this range2, and there was no clear dose-related trend. While few other changes in clinical biochemistry parameters were statistically significant (higher glucose values in main group males at 100 mg/kg bw/day, higher calcium and lower phosphate values in main group males at 300 mg/kg bw/day, lower protein and albumin values in main group females, higher chloride and potassium levels in females at the end of the recovery period), the alterations in these clinical biochemistry parameters were considered unrelated to administration of the test item due to the minimal magnitude of the change, absence of a dose response, absence of similar changes at the end of treatment and/or absence of correlating histological findings.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were not considered to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
A slightly lower hind-limb grip strength (not statistically significantly) was noted for the Main and Recovery females at 1000 mg/kg bw/day at the end of treatment. Mean hind-limb strength at 1000 mg/kg bw/day remained within the range considered normal for rats of this age and strain. Also, there was no apparent trend towards a decrease based on individual values across the dose groups. Therefore, these variations were not considered to represent an effect of treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations. At 100 mg/kg bw/day, one male (no. 22) showed a marked abscess of the muscular wall of the stomach with central foreign material (hair) and a female (no. 68) showed at necropsy a constriction of one uterine horn (the microscopic correlate was dilation of the horn and vacuolation of the epithelium). At 1000 mg/kg bw/day, a transitional cell papilloma of the urinary bladder of one female (no. 95) was noted. This benign neoplasm with exophytic growth pattern of fibrovascular stalks lined by regular transitional epithelium, occurred at a single incidence and was not accompanied by other urinary bladder changes in any other animal and can occasionally be observed in control rats. All of the above findings were regarded as unrelated to treatment with the test-item. 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.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects on T4 levels in Main males.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were not considered to have been affected by treatment.
Most females had regular cycles of 4 days. Two females at 100 mg/kg bw/day (nos. 70 and 74; both delivered offspring), two females at 300 mg/kg bw/day (nos. 82 (delivered offspring) and 84 (not pregnant) and two females at 1000 mg/kg bw/day (nos. 91 (implantation sites only) and 92 (not pregnant)) had an irregular cycle at pretest and/or premating. In absence of a dose-related incidence, these findings did not indicate a relation with treatment.

Description (incidence and severity):

There were 1/10 couples of the 300 mg/kg bw/day group and 2/10 couples of the 1000 mg/kg bw/day group with no offspring. There were no abnormalities seen in the reproductive organs of these rats, which could account for their lack of offspring. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item. Stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis and qualitative assessment of the ovaries did not reveal any abnormalities.
Mating index was not affected by treatment. All females showed evidence of mating.
Precoital time was not affected by treatment. All females showed evidence of mating within 4 days
Number of implantation sites were comparable in all groups and within the expected range. One female at 1000 mg/kg bw/day (no. 91; no offspring) had two implantation sites only, resulting in a marginally lower mean number of implantation sites at this dose. This was an incidental occurrence and the mean remained within the range considered normal for rats of this age and strain3. As such, this variation was not considered to be related to treatment.
Fertility index was not considered to be affected by treatment. One female at 300 mg/kg bw/day (no. 84) and one female at 1000 mg/kg bw/day (no. 92) were not pregnant. Since these cases of non-pregnancy showed no dose-related incidence across the dose groups, occurred in absence of any reproductive/developmental toxicity and remained within the range considered normal for rats of this age and strain3, this was not considered to be related to treatment.
Gestation index and duration of gestation were affected by treatment. One female at 1000 mg/kg bw/day (no. 91) had implantation sites only. This incidence remained within the range considered normal for rats of this age and strain. Also, given its incidental occurrence, this was not considered to be related to treatment.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were related to treatment. The nature and incidence of observed clinical signs remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment. Three pups at 300 mg/kg bw/day (all of litter no. 78) were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was not affected by treatment. One pup of the control group (litter no. 56) and two pups at 1000 mg/kg bw/day (litter 95) were missing or found dead on PND 2 or 3. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was not affected by treatment. No pups were found dead/missing between lactation Days 5 and 13.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights of pups were not considered to be affected by treatment. The statistically significantly higher mean body weight for female pups at 1000 mg/kg bw/day on Day 1 remained within the range considered normal for rats of this age and strain4 and no trend was apparent regarding dose and/or duration of lactation phase. This variation was therefore considered to have arisen by chance and not considered to be related to treatment.

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):

Serum T4 levels in male and female PND 13-15 pups were comparable in all groups.

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was not considered to be affected by treatment.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No compound-related macroscopic findings were noted among pups. The nature and incidence of macroscopic findings remained within the range considered normal for pups of this age, and were therefore not considered to be related to treatment.

Histopathological findings:

not examined

The total number of offspring born compared to the total number of uterine implantations was not affected by treatment. For female nos. 56 (control), 68 (100 mg/kg bw/day), 83 (300 mg/kg bw/day) and 95 (1000 1000 mg/kg bw/day), the number of pups was slightly higher than the number of implantations. This phenomenon is observed from time to time and is caused by normal resorption of these areas during the 14 days of lactation. No toxicological relevance was attached to this finding in the current study.
Litter size was not affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to 1000 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Reproductive effects observed:

no

Table 4. Reproductive Performance

Group	Dose level mg/kg bw/day	Female/Male No.	In-Life Reason	Histopathology
1	0	-	-	-
2	100	-	-	-
3	300	84/34	Not pregnant	No abnormalities noted
4	1000	91/42	No offspring	No abnormalities noted
		92/43	Not pregnant	No abnormalities noted

Table 5. Reproduction Data Summary

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	100 MG/KG	300 MG/KG	1000 MG/KG
Females paired	10	10	10	10
Females mated	10	10	10	10
Pregnant females	10	10	9	9
Females with implantations only	0	0	0	1
Females with living pups on Day 1	10	10	9	8
Mating index (%) (Females mated / Females paired) * 100	100	100	100	100
Fertility index (%) (Pregnant females / Females mated) * 100	100	100	90	90
Gestation index (%) (Females with living pups on Day 1 / Pregnant females) * 100	100	100	100	89

Table 6. Implantation Sites Summary

		GROUP 1	GROUP 2	GROUP 3	GROUP 4
		CONTROL	100 MG/KG	300 MG/KG	1000 MG/KG
NECROPSY Implantations	MEAN	13.2	12.2	12.6	11.1
	ST.DEV	2.1	3.6	1.6	3.8
	N	10	10	9	9

Table 7. Developmental Data

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	100 MG/KG	300 MG/KG	1000 MG/KG
LITTERS TOTAL	10	10	9	8
DURATION OF GESTATION
MEAN (+)	21.2	21.4	21.3	21.4
ST.DEV.	0.4	0.5	0.7	0.5
N	10	10	9	8
DEAD PUPS AT FIRST LITTER CHECK
LITTERS AFFECTED (#)	0	0	1	0
TOTAL	0	0	3	0
MEAN (+)	0	0	0.3	0
ST.DEV.	0	0	1	0
N	10	10	9	8
LIVING PUPS AT FIRST LITTER CHECK
% OF MALES / FEMALES (#)	49 / 51	50 / 50	52 / 48	42 / 58
TOTAL	123	111	100	89
MEAN (+)	12.3	11.1	11.1	11.1
ST.DEV.	1.9	2.9	2.5	2.1
N	10	10	9	8
POSTNATAL LOSS
% OF LIVING PUPS	0.8	0	0	2.2
LITTERS AFFECTED (#)	1	0	0	1
TOTAL (#)	1	0	0	2
MEAN (+)	0.1	0	0	0.3
ST.DEV.	0.3	0	0	0.7
N	10	10	9	8
CULLED PUPS
TOTAL	42	34	30	23
LIVING PUPS DAY 4 P.P.
TOTAL	80	77	70	64
MEAN (+)	8	7.7	7.8	8
ST.DEV.	0	0.9	0.7	0
N	10	10	9	8
BREEDING LOSS DAYS 5 - 13 P.P.
% OF LIVING PUPS AT DAY 4 P.P.	0	0	0	0
LITTERS AFFECTED (#)	0	0	0	0
TOTAL (#)	0	0	0	0
MEAN (+)	0	0	0	0
ST.DEV.	0	0	0	0
N	10	10	9	8
LIVING PUPS DAY 13 P.P.
% OF MALES / FEMALES (#)	51 / 49	49 / 51	53 / 47	53 / 47
TOTAL	80	77	70	64
MEAN (+)	8	7.7	7.8	8
ST.DEV.	0	0.9	0.7	0
N	10	10	9	8

+/++ Steel-test significant at 5% (+) or 1% (++) level

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

Table 8. Developmental Data (contd.)

	GROUP 1	GROUP 2	GROUP 3	GROUP 4
	CONTROL	100 MG/KG	300 MG/KG	1000 MG/KG
Total number of offspring born	123	111	103	89
Total number of uterine implantation sites	132	122	113	100
Number of live offspring on Day 1 after littering	123	111	100	89
Number of live offspring on Day 4 (before culling)	122	111	100	87
Number of live offspring on Day 4 (after culling)	80	77	70	64
Number of live offspring on Day 13 after littering	80	77	70	64
Post-implantation survival index (%)	93	91	91	89
Live birth index (%)	100	100	97	100
Viability index (%)	99	100	100	98
Lactation index (%)	100	100	100	100

Conclusions:

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with a 14-day recovery period, the following no-observed-adverse-effect level (NOAEL) was established: at least 1000 mg/kg bw/day.

Executive summary:

The objectives of this study were to determine the potential toxic effects of the test item when given orally by gavage for a minimum of 28 days to Wistar Han rats, followed by a 14-day recovery period and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. The recovery animals (used to study the potential reversibility of possible toxic effects) were not mated and consequently were not used for the assessment of reproduction/ developmental toxicity. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated. The dose levels in this study were selected to be 0, 100, 300 and 1000 mg/kg bw/day, based on the results of the dose range finder. Ten animals per sex and dose were used, additional five animals per sex were used in control and high dose groups for the recovery examinations.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment), body weight and food consumption, estrous cycle determination, clinical pathology (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment and end of recovery), measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, 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 13-15 pups)). Formulation analyses confirmed that formulations of test item in corn oil were prepared accurately and homogenously. No parental toxicity was observed up to 1000 mg/kg bw/day. No reproductive toxicity was observed up to 1000 mg/kg bw/day. No developmental toxicity was observed up to 1000 mg/kg bw/day. In conclusion, based on the results of this combined 28-day repeated dose toxicity study with

the reproduction/developmental toxicity screening test with a 14-day recovery period, the following no-observed-adverse-effect levels were established for the test item:

Parental NOAEL: at least 1000 mg/kg bw/day

Reproduction NOAEL: at least 1000 mg/kg bw/day

Developmental NOAEL: at least 1000 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via dermal route

Endpoint conclusion:

no adverse effect observed

Additional information

In a GLP-compliant 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test (according to OECD guideline 422), Wistar Han rats were treated with the test article by daily oral gavage at dose levels of 100, 300 or 1000 mg/kg bw/day, followed by a 14-day treatment-free recovery period. The rats of the control group received the vehicle, corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 13-15 days of lactation (for 50-56 days). Females that failed to deliver pups were treated for 42 days. Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels. The following parameters and end points were evaluated in this study: mortality/ moribundity, clinical signs, functional observations (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment), body weight and food consumption, estrous cycle determination, clinical pathology (for 5 selected animals/sex/group and all Recovery group animals at the end of treatment and end of recovery), measurement of thyroid hormone T4 (F0-males), gross necropsy findings, organ weights and histopathologic examinations. In addition, the following reproduction/developmental parameters were determined: mating and fertility indices, 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 13-15 pups)).

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the parameters investigated in this study. No reproductive toxicity was observed up to 1000 mg/kg bw/day. No developmental toxicity was observed up to 1000 mg/kg bw/day. In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test with a 14-day recovery period, the following no-observed-adverse-effect levels were established for the test item:

Parental NOAEL: at least 1000 mg/kg bw/day

Reproduction NOAEL: at least 1000 mg/kg bw/day

Developmental NOAEL: at least 1000 mg/kg bw/day

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item revealed no adverse findings regarding fertility and development in a screening assay. As a result the substance is not classified for reproduction and developmental toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.