Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

No reproduction toxicity (fertility) was observed up to the highest dose level (1000 mg/kg bw/day) orally tested in rats according to OECD TG 422 (van de Ven, 2018). For the endpoint toxicity to reproduction no one- or multigeneration reproductive toxicity studies are available. Data waiver is claimed.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Aug to Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl: WI(Han)
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. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) Males: 10 weeks; Females: 13 weeks
- Weight at study initiation: (P) Males: 295-322 g; Females: 194-250 g
- 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 Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, 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 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.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: dried corn oil
Details on exposure:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. Until 6 Oct 2017, the dosing formulations were prepared daily as a solution and dosed within 2 hours after completion of the preparation of the test item. From 7 Oct 2017 (Day 24 of treatment) onwards, dosing formulations were prepared in daily portions as a solution and dosed within 24 hours after completion of the preparation of the test item, as stability was confirmed over a period of 24 hours at room temperature.

Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations 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. Any residual volumes were discarded.
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.

Since less than 9 females in Group 4 have shown evidence of mating after 14 days of cohabitation, each female without evidence of mating (nos. 42 and 73 of Groups 1 and 4, respectively) was re-mated with a male of proven fertility of the same group for one day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentration Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ±10 % for solutions of target concentration.

- Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was <=10 %.

- Stability Analysis
During the course of this study at one occasion during the treatment phase, stability of the prepared formulation was determined at 24 hours at room temperature. Duplicate sets of each sample (approximately 500 mg) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ±10 % of the concentration determined by the initial analysis of each formulation.
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 29 days. Males were treated for 29 days (most males) or 33 days (two males used for re-mating), up to and including the day before scheduled necropsy. This included a minimum of 14 days prior to mating and during the mating period. Females that delivered were treated for 50-64 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 13-15 days after delivery, up to and including the day before scheduled necropsy. Females which failed to deliver were treated for 42 or 53 days.

Female nos. 45, 48 (Group 1), 51, 55 and 60 (Group 2) were not dosed on one occasion as these females were littering at the moment of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Frequency of treatment:
Once daily at approximately the same time each day.
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
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale
The dose levels were selected based on the results of a 10-day dose range finder with oral administration of 500 and 1000 mg/kg test item in the rat (Test Facility Study No. 518884) and in an attempt to produce graded responses to the test item.

Based on the results of the dose range finder, selected dose levels for the main study were 100, 300 and 1000 mg/kg. The peak effect of occurrence of clinical signs occurred at 3 hours after dosing. This time point was taken into account to set a time range within which clinical observations and functional observation tests were conducted after dosing in the main study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: 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.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed 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 3 hours (±30 min) post-dose.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated 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. In order to monitor the health status, female no. 74 (Group 4) was also weighed on Day 6 of the mating period. Female no. 49 (Group 1) was also weighed on Day 25 post-coitum in order to monitor the pregnancy status. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was quantitatively measured weekly, 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.

FUNCTIONAL TESTS: Yes
- Time schedule for examinations: Functional tests (hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity) were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 11-13). These tests were performed 3 hours (±30 min) post-dose, after completion of clinical observations.

CLINICAL PATHOLOGY: Yes
Blood of F0-animals (except for animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. When non-serum samples were clotted, additional blood samples were obtained in the necropsy room (if possible). After collection all samples were transferred to the appropriate laboratory for analysis. F0-females were not fasted prior to blood sampling and necropsy. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available.
- Hematology parameters: White blood cells (WBC), Red Blood Cell Distribution Width (RDW), Neutrophil (absolute), Haemoglobin, Lymphocyte (absolute), Haematocrit, Monocyte (absolute), Mean corpuscular volume (MCV), Eosinophil (absolute), Mean corpuscular haemoglobin (MCH), Basophil (absolute), Mean corpuscular haemoglobin concentration (MCHC), Red blood cells, Platelet, Reticulocyte (absolute).
- Coagulation parameters: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)
- Clinical chemistry parameters: Alanine aminotransferase (ALAT), Creatinine, Aspartate aminotransferase (ASAT), Glucose, Alkaline Phosphatase (ALP), Cholesterol, Total protein, Sodium, Albumin, Potassium, Total Bilirubin, Chloride, Bile Acids, Calcium, Urea, Inorganic Phosphate (Inorg. Phos).
- Thyroid hormone: Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4). Measurement of T4 was conducted for F0-males. For the F0-generation, assessment of T4 (females) and Thyroid Stimulating Hormone (TSH; both sexes) was considered not relevant because no toxicologically relevant changes were noted in T4 in F0-males or in the weight and morphology of the thyroid of F0-animals of both sexes.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage. Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
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.
Litter observations:
Pups were observed daily for general health/mortality. The number of live and dead pups were determined on PND 1 and daily thereafter. Clinical observations were performed at least once daily for all pups. Live pups were weighed individually on PND 1, 4, 7 and 13. Sex was externally determined for all pups on PND 1 and 4. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13. 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.
Postmortem examinations (parental animals):
SACRIFICE
- 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: Males (which sired and failed to sire): Following completion of the mating period (a minimum of 28 days of administration);
Females which delivered: PND 14-16; Females which failed to deliver: With evidence of mating: Post-coitum Day 26-27 (Nos. 49, 65, 69 and 75). Without evidence of mating: 25 days after the last day of the mating period (No. 46).

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs. The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, 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
- Representative samples of the tissues identified in the tables 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.

- Table 1: Tissue Collection and Preservation for all selected animals, and all animals that died spontaneously or were sacrificed in extremis: Animal identification, artery, aorta, body cavity, nasopharynx, bone marrow, bone (femur and sternum), brain (seven levels), cervix, epididymis, esophagus, eye, adrenal gland, coagulation gland, harderian gland, lacrimal gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, salivary gland, seminal vesicle gland, thyroid gland, gross lesions/masses, gut-associated lymphoid tissue, heart, kidney, large intestine (cecum, colon, rectum), larynx, liver, lung, lymph node (mandibular and mesenteric site), skeletal muscle, optic nerve, sciatic nerve, ovaries, pancreas, skin, small intestine (duodenum, ileum, jejunum), spinal cord,spleen, stomach, testes, thymus, tongue, trachea, urinary bladder, uterus, vagina.

- Table 2: Tissue Collection and Preservation for all remaining animals (incl. males that failed to sire females that failed to deliver pups; non-pregnant, implantation sites only):
Animal identification, cervix, epididymis, coagulation gland, mammary gland, parathyroidc gland, pituitary gland, prostate gland, seminal vesicle gland, thyroid gland, gross lesions/masses, ovaries, testes, uterus, vagina.
Postmortem examinations (offspring):
SACRIFICE
- Method of Euthanasia: Pups, younger than 7 days were euthanized by decapitation. All remaining pups (PND14-16), except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital (Euthasol® 20 %). The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.
- Unscheduled Deaths: Pups that died before scheduled termination were examined externally and sexed (both externally and internally). Pups found dead during the weekend were fixed in identified containers containing 70 % ethanol as they were not necropsied on the same day. 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.
- Scheduled Euthanasia: 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 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. 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) was preserved in 10 % buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
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 3 observations.

The following pairwise comparisons were made: Group 2 vs. Group 1, Group 3 vs. Group 1 and Group 4 vs. Group 1.

Parametric: Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric: 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.

Incidence: 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 index (%): Number of females mated / Number of females paired x 100

Precoital time: Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): Number of pregnant females / Number of females mated x 100

Gestation index (%): Number of females with living pups on Day 1 / Number of pregnant females x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): Total number of offspring born / Total number of uterine implantation sites x 100

Live birth index (%): Number of live offspring on Day 1 after littering / Total number of offspring born x 100

Percentage live males at First Litter Check (%): Number of live male pups at First Litter Check / Number of live pups at First Litter Check x 100

Percentage live females at First Litter Check (%): Number of live female pups at First Litter Check / Number of live pups at First Litter Check x 100

Viability index (%): Number of live offspring on Day 4 before culling / Number live offspring on Day 1 after littering x 100

Lactation index (%): Number of live offspring on Day 13 after littering / Number live offspring on Day 4 (after culling) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations. Salivation seen in animals of all groups, most frequently at 300 and 1000 mg/kg, was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing). Any other clinical signs noted incidentally 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 and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test item. There were two premature deaths, both due to gavage related trauma (female nos. 54 and 74 of the 100 and 1000 mg/kg groups, respectively). These deaths were considered not to be test item-related.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related changes in clinical chemistry parameters. The mean concentration of bile acids appeared higher in males at 1000 mg/kg. The difference from controls was not statistically significant and could largely be explained by the high value in male no. 31 (value of 81.3 μmol/L). In the absence of associated adverse anatomic pathology changes, this isolated finding was considered not to represent an adverse effect of the test item. Isolated statistically significant variations noted in clinical chemistry parameters were considered unrelated to treatment due to the lack of a dose-related trend.

Thyroid hormone analyses: Serum levels of T4 in F0 males were not affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically relevant alterations in organ weights. Compared to the control group, mean thyroid weights (absolute and relative to body weight) appeared somewhat higher in all groups of treated males, reaching statistical significance at 1000 mg/kg). The relative differences showed no dose-related trend. These changes were considered to have (partly) arisen as a result of slightly low control values, particularly in control male no. 3 for which a reduced size of the thyroid was noted at necropsy. Individual relative thyroid weight of the majority of the treated males was similar to the historical control mean. In the absence of any test item-related macroscopic or microscopic finding in the thyroid gland this increase in thyroid gland weight was considered non-adverse. The statistically significantly higher uterus weights (absolute and relative to body weight) noted in 100 mg/kg females were considered unrelated to treatment due to the lack of a dose-related trend.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no test item-related microscopic observations. All 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.

Reproductive performance: A few couples had no offspring: female/male nos. 46/6 and 49/9 of the control group (female no. 46 was not mated, no. 49 had implantations only); nos. 65/25 and 69/29 at 300 mg/kg (both females were not pregnant); nos. 75/35 and 74/34 at 1000 mg/kg (female 75 was not pregnant, no. 74 was euthanized in extremis on Day 6 of the mating period and showed no evidence of mating). In addition, two males (nos. 2 and 33 of the control and 1000 mg/kg groups, respectively) had not mated during 14 days of cohabitation. Histopathology did not reveal any changes in the reproductive organs that could explain these cases of reproductive failure.

There were no remarkable findings in the reproductive organs of the 100 mg/kg male (no. 14) which was not used for pairing due to the premature death of the pertaining female (no. 54). There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment. Most females had regular cycles of four or five days. Extended di-estrus during pairing occurred in three control females (nos. 42, 44 and 46), two females at 300 mg/kg (nos. 68 and 69) and two females at 1000 mg/kg (nos. 73 and 79). Except for nos. 46 and 69, these females had live offspring. As the incidence of this finding was within normal limits and showed no dose-related trend, this was considered to be unrelated to treatment.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was considered not to be affected by treatment. Except for one control female (no. 46) and one 1000 mg/kg female (no. 74, euthanized in extremis on Day 6 of the mating period), all females showed evidence of mating. The mating indices were 90 % for the control and 1000 mg/kg groups and 100 % for the other groups. Two males (nos. 2 and 33 of the control and 1000 mg/kg groups, respectively) had not mated during 14 days of cohabitation (the pertaining females mated successfully upon re-mating with another male). These cases of unsuccessful mating were not supported by morphological changes in the male or female reproductive organs.

Precoital time was considered not to be affected by treatment. Most females showed evidence of mating within five days.

Number of implantation sites was not affected by treatment.

Fertility index was considered not to be affected by treatment. Except for two females at 300 mg/kg (nos. 65 and 69) and one female at 1000 mg/kg (no. 75), all mated females were pregnant. These cases of non-pregnancy, all without related histopathology changes in reproductive organs, were considered to be unrelated to treatment as their incidence showed no dose-related trend and were within normal limits. The fertility index was 100, 100, 80 and 89 % for the control, 100, 300 and 1000 mg/kg groups, respectively.

Gestation index and duration of gestation were not affected by treatment. Except for one female of the control group (no. 49), all pregnant females had live offspring. The gestation indices were 89 % for the control group and 100 % for the other groups. This incidental case of failed pregnancy in the control group occurred without related histopathology changes in reproductive organs (the female had two implantation sites only).

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.

Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices
were 83, 97, 93 and 92 % for the control, 100, 300 and 1000 mg/kg groups, respectively. It was noted that the index for the control group was lower than normal. This was due to the high post-implantation loss in female no. 44 which lost 8/14 implantations. For female no. 76 of the 1000 mg/kg group the number of pups born was slightly higher than
the number of implantation sites. This phenomenon is observed from time to time and is caused by normal resorption of these areas during lactation.

Litter size was considered not affected by treatment.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices were 99 % for the 300 mg/kg group and 100 % for the other groups. At first litter check, one pup at 300 mg/kg (litter no. 61) was found dead. This incidental pup mortality was considered unrelated to treatment.

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99 % for the 100 mg/kg group and 100 % for the other groups. The incidental death of one pup (litter no. 59) at 100 mg/kg which went missing (presumably cannibalized) on PND 4 was considered unrelated to treatment.

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. No pups died after PND 4, resulting in a lactation index of 100 % for all groups.
Dose descriptor:
NOAEL
Remarks:
general toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related effects observed
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. The clinical signs observed remained within the range considered normal for pups of this age and the incidence showed no dose-related trend. These findings were therefore considered to be unrelated to treatment.
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment. The nature and incidence of macroscopic findings noted incidentally remained within the range considered normal for pups of this age, and were therefore judged to be unrelated to treatment.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
- Sex ratio
Sex ratio was considered not to be affected by treatment.

- Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment.

- Areola/nipple retention
Treatment up to 1000 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

- Clinical biochemistry (T4 levels)
There were no treatment-related changes in serum T4 levels in male and female PND 14-16 pups.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
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 treatment-related effects observed
Critical effects observed:
no
Reproductive effects observed:
no
Executive summary:

The objectives of this study were to determine the potential toxic effects of 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type (IPDI oligomers, allophanate type) when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

Ten male and ten female Wistar Han rats per group were treated with IPDI oligomers, allophanate type by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg according to OECD TG 422. The rats of the control group received the vehicle, dried corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (29 to 33 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (50 to 64 days). Females that failed to deliver pups were treated for 42 or 53 days.

Formulation analysis showed that the formulations were prepared accurately and homogeneously, and that the formulations were stable for at least 24 hours at room temperature under normal laboratory light conditions.

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). Treatment with the test item up to 1000 mg/kg was well tolerated as indicated by the absence of adverse changes in the parental parameters and examinations in this study (i.e. clinical appearance, body weight, food consumption, functional tests, haematology and clinical chemistry, macroscopic examination, organ weights, and microscopic examination). The only treatment-related finding in this study consisted of slight salivation after dosing, noted most frequently at 300 and 1000 mg/kg. This was regarded as a physiological response rather than a sign of systemic toxicity.

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopy).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of IPDI oligomers, allophanate type were established:

Parental NOAEL: at least 1000 mg/kg

Reproduction NOAEL: at least 1000 mg/kg

Developmental NOAEL: at least 1000 mg/kg

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is GLP compliant and is of high quality (Klimisch score=1)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Ten male and ten female Wistar rats per group were treated with 3 -Isocyanatomethyl-3,5,5 -trimethylcyclohexyl isocyanate, oligomers, allophanate type (IPDI oligomers, allophanate type) by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg according to OECD TG 422 (van de Ven, 2018). The rats of the control group received the vehicle, dried corn oil, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (29 to 33 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13-15 days of lactation (50 to 64 days). Females that failed to deliver pups were treated for 42 or 53 days. The objectives of this study were to determine the potential toxic effects of IPDI oligomers, allophante type when given orally by gavage for a minimum of 28 days to Wistar rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

Parental results: No parental toxicity was observed up to the highest dose level tested (1000 mg/kg). Treatment with the test item up to 1000 mg/kg was well tolerated as indicated by the absence of adverse changes in the parental parameters and examinations in this study (i.e. clinical appearance, body weight, food consumption, functional tests, haematology and clinical chemistry, macroscopic examination, organ weights, and microscopic examination). The only treatment-related finding in this study consisted of slight salivation after dosing, noted most frequently at 300 and 1000 mg/kg. This was regarded as a physiological response rather than a sign of systemic toxicity.

Reproductive results: No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Developmental results: No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopy).

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of IPDI oligomers, allophanate type were established: Parental NOAEL: at least 1000 mg/kg, Reproduction NOAEL: at least 1000 mg/kg and Developmental NOAEL: at least 1000 mg/kg.

No one- or multigeneration reproductive toxicity studies are available for IPDI oligomers, allophanate type. A data waiver is claimed for this type of study. Due to the lack of adverse effects on reproductive organs in male and female rats up to an oral dose of 1000 mg/kg bw/day in a OECD 422 study (van de Ven, 2018) or up to an inhalation exposure of 122 mg/ m³ in a subacute toxicity study (Pauluhn, 2012), there is no indication of a specific toxic potential to the reproductive organs. Therefore based on the considerations above and in accordance with Annex IX of REACH Regulation (EC) No 1907/2006 at this stage no further testing by means of an extended one-generation reproductive toxicity study (EOGRTS) is required for the fertility assessment of IPDI oligomers, allophanate type.

Effects on developmental toxicity

Description of key information

No developmental toxicity (teratogenicity) was observed up to the highest dose level (1000 mg/kg bw/day) orally tested in rats according to OECD TG 414 (LPT, 2017b). For that endpoint no prenatal developmental toxicity study (OECD TG 414) in a second species (rabbit) was performed. Data waiver is claimed.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is GLP compliant and is of high quality (Klimisch score=1)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

No studies on developmental toxicity are performed with 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, allophanate type (IPDI oligomers, allophanate type). Therefore, for the endpoint developmental toxicity a rat prenatal developmental toxicity study with the structurally similar 3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate homopolymer, isocyanurate type (IPDI oligomers, isocyanurate type) was used via read-across. IPDI oligomers, isocyanurate type is also an IPDI-based polyisocyanate and is, moreover, contained to approx. 50 % in IPDI oligomers, allophanate type. The developmental toxicity study (OECD TG 414) with IPDI oligomers, isocyanurate type  revealed no substance-related findings for the fetuses and the dams at all after oral administration up to 1000 mg/kg bw through day 6 to 20 of gestation (LPT, 2017b). The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were also not influenced by the test item. Therefore, the no-observed-adverse-effect level (NOAEL) for the fetal organism was 1000 mg test item/kg bw/day (for additional information see justification for the read-across approach to IPDI oligomers, allophanate type attached to this endpoint summary).

Additionally, three fully reliable inhalation developmental toxicity studies on rats according to OECD TG 414 are available based on read-across for comparable aliphatic isocyanates (monomeric IPDI, H12MDI, HDI) and give no indication for any specific developmental toxicity for the aliphatic isocyanates. Fetal development is affected only at levels that causes clear maternal toxicity and thus considered as secondary effect (Klaus, 2004; Langewische, 2004; Astroff, 2000). A comprehensive discussion on the mode of action, general toxicity and reproductive/developmental toxicity of aliphatic isocyanates is attached to this endpoint summary (see ALIPA_Waiving_Reproductive_Toxicity_Final_2009-11-18). Based on this document a conclusion is drawn that none of the aliphatic isocyanates is a developmental toxicant.

For the endpoint developmental toxicity no prenatal developmental toxicity study (OECD TG 414) in a second species (rabbit) was performed since no indication of prenatal developmental toxicity was observed in a first prenatal developmental toxicity study (OECD TG 414) on rats and no triggers for adverse effects on the development of rats could be detected in repeated dose toxicity studies (OECD TG 412, 413, 422) up to the highest dose levels after oral or inhalation exposure. Therefore, a data waiver is claimed.

Justification for classification or non-classification

According to Regulation (EC) No 1272/2008, Annex I, no classification is warranted for toxicity to reproduction.