Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
acc. to OECD TG 422
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
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

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: Males: 10 weeks; Females: 13 weeks
- Weight at study initiation: 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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Application volume: 5 mL/kg
Vehicle:
other: dried corn oil
Details on oral 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.
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.
Doses / concentrationsopen allclose all
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 IPDI oligomers, allophanate type 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.

Examinations

Observations and examinations performed and frequency:
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.
Sacrifice and pathology:
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.
Other examinations:
For examinations on reproductive function/performance of parental animals as well as on developmental toxicity of pups see section 7.8.1.
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.

Results and discussion

Results of examinations

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.

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

Effect levels

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 treatment-related effects observed

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Executive summary:

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.

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.

In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental NOAEL of IPDI oligomers, allophante type was at least 1000 mg/kg bw.