Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

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

Study period:

From January 17, 2017 to March 05, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The dose levels were selected based on available acute oral toxicity data (LD50 > 2000 mg/kg bw in rats and based on the results of the Dose Range Finding (DRF) study. The aim was to induce toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose level. During the Dose Range Finding study, no treatment related adverse effects were seen at dose levels of 100, 300 or 1000 mg/kg bw/day.

Specific details on test material used for the study:

CAS No.: 162492-07-1; EC No.: 500-740-9; Batch No.: 1591ZG-101; Purity: ca. 100 %; Appearance: white fine powder

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for dose range finding study.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:WI Wistar rats
Source: Charles River Laboratories, Research Models and Services, Germany, from SPF colony
Hygienic conditions: standard laboratory conditions
Number of animals: 48 male, 48 female rats, 12 animals/sex/group, 4 groups
Age of animals: young adult rats, approximately 10 weeks old at start and 12 weeks old at mating
Body weight range: males: 408-443 g, females: 240-267 g; did not exceed ±20 % of the mean weight for each sex at onset of treatment
Acclimation period: 5 days

Husbandry
Animal health: only healthy animals were used for the test, as certified by the clinical veterinarian. Females were nulliparous and non-pregnant.
Cage type: type II polycarbonate
Bedding: LIGNOCEL ¾ S certified wooden chips produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Nesting: Arbocel nest building material produced by J. Rettenmaier & Söhne GmbH+Co.KG, Germany
Light: 12 hours daily
Temperature: 19.0 – 24.8 °C
Relative humidity: 24 – 69 %
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: rodents were group-housed, up to 4 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively

Food and water supply
Animals received ssniff SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, Germany, ad libitum, and tap water from the municipal supply from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each parental/adult animal (P Generation) was identified by a unique number within the study, written with indelible ink on the tail.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

The test substance was formulated in the vehicle with a pestle and mortal or with a “Ultra Turrax T-25” mixer. Formulations were prepared daily. The dosing solutions were administered to the test substance or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.

Details on mating procedure:

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male from the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 5 days. A vaginal smear was prepared daily during the mating period and stained with 1 % aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test substance formulations for concentration and homogeneity was performed using a validated LC-MS Method. Top, middle and bottom duplicate samples were taken and analysed from test substance formulations on 3 occasions. One set was collected for analysis and one set as a back-up, if required for any confirmatory analyses. Similarly, one sample was taken on each occasion in duplicate from the middle of the vehicle control solution (Group 1) to confirm the absence of test substance.

Duration of treatment / exposure:

Dosing of both sexes began after 5 days acclimatisation and 2 weeks before mating, during the mating, and were continued up to and including the day before the necropsy.
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination.
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (4-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum (PPD 0).

Frequency of treatment:

daily

Details on study schedule:

Dosing scheme, Male animals:
Acclimatisation: 5 days
Pre-mating period: 14 days
Mating/Post-mating period: at least 14 days (Last week of treatment: FOB, Day 24, 5 animals/group, Prior to/at necropsy examinations

Dosing scheme, Female animals:
Acclimatisation period: 5 days
Pre-mating period: 14 days
Mating: up to 5 days
Gestation: 22-24 days
Delivery
Lactation period: at least 4 days, FOB: (PND 4, 5 animals/group); Pups necropsy, (PND 4)
PND 5: Prior to/at necropsy examinations

All F1 offspring were terminated on Day 4 post-partum. In order to allow for overnight fasting of dams prior to urine collection on PPD 5, the offspring were euthanized on PPD/PND 4 and the dams on PPD/PND 5.

Dose / conc.:

0 mg/kg bw/day

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Details on study design:

Observation of the delivery process, offspring and nursing instinct:

Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.
Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.
Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0 or 1) and on PND 4, with accuracy of 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded. All pups were culled on PND 4.

Parental animals: Observations and examinations:

The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals

Sperm parameters (parental animals):

Special attention was paid to the evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Litter observations:

The viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4.

Postmortem examinations (parental animals):

Organ weight measurements:
At the time of termination, body weight and the weight of the following organs from all adult animals were determined:
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually.

For the adult animals, a detailed histological examination was performed as follows:
- on the selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group),
- all macroscopic findings (abnormalities), except of minor order from all animals,
- retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and additionally of the one male that failed to sire and the one female that failed to deliver healthy pups.

Postmortem examinations (offspring):

Macroscopic Findings
On PND 0, there were 3 Low Dose and 9 Mid Dose pups found dead that remained intact (not cannibalized or autolysed). Those were subjected to necropsy with macroscopic examination. No test substance-related macroscopic findings were seen. No other pups that died during the lactation period could be examined for test substance-related macroscopic findings.

Organ weights
Compared to the control, there were no statistically significant differences in mean values recorded in any of the dose groups.

Statistics:

Data was recorded on the appropriate forms from the relevant SOPs of the labolatory and then tabulated using the Microsoft Office Word and/or Excel, or using the software PROVANTIS v.9, as appropriate. Numerical data obtained during the conduct of the study was subjected as appropriate to calculation of group means
and standard deviations.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related clinical signs were observed during the study. One female animal in the Low dose group had piloerection on Day 41. Based on the very low incidence and lack of dose response, this observation was considered to be incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

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):

Significantly higher sodium (p<0.01), chloride (p<0.01), total protein (p<0.05) and bile acid (p<0.05) concentrations were recorded for High dose (1000 mg/kg bw/day) males. No such things were seen in females. These findings are within the historical control range. Due to the incidental appearances and the lack of any supporting evidence of any changes in these animals (histopathology, urinalysis, etc.) and as the recorded values are within the historical control range, these differences were considered to not reflect any adverse effects of the test substance.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance-related effects observed at any dose

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

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:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Histopathological findings:

effects observed, non-treatment-related

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No substance-related effects observed at any dose

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

All the dose formulations were homogenous. The measured concentrations of the test substance evaluated for each test substance-dose group varied between 91 % and 105 % of the nominal contents. No test substance was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.

Conclusions:

Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male parental/adult generation, and also for the F1/pups generation.

Executive summary:

A study was conducted to determine the reproductive/developmental toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. The screening test intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-partum Day (PPD) 4. The details on systemic effects are summarised in section ‘Repeated dose toxicity: oral’. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This corresponded to 28 d in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PPD) 4. The test substance, formulated in corn oil, was administered by oral gavage to test animals. One control group and three treated groups were tested (100, 300 and 1000 mg/kg bw), each consisting of 12 males and 12 females. The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 5. There were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male parental/adult generation, and also for the F1/pups generation (Weisz, 2017).

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 study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Toxicity to reproduction

A study was conducted to determine the reproductive/developmental toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. The screening test intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-partum Day (PPD) 4. The details on systemic effects are summarised in section ‘Repeated dose toxicity: oral’. Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This corresponded to 28 d in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PPD) 4. The test substance, formulated in corn oil, was administered by oral gavage to test animals. One control group and three treated groups were tested (100, 300 and 1000 mg/kg bw), each consisting of 12 males and 12 females. The reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until post-natal Day (PND) 4. No test substance related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD 5. There were no adverse effects on the F1 offspring viability, clinical signs, development or at observations following euthanasia. Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male parental/adult generation, and also for the F1/pups generation (Weisz, 2017).

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of a combined 28 d oral toxicity study with a reproduction/developmental toxicity screening, the test substance does not need to be classified for reproductive toxicity according to CLP (EC 1272/2008) criteria.