Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

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

Justification for type of information:

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.

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

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

Details on route of administration:

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.

Vehicle:

olive oil

Details on oral 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. Male and female rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day (PPD) 4.

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:

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).

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent no treatment

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FUNCTIONAL OBSERVATION BATTERY (FOB) for all animals:

Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day.
General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment.
On gestation Day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. More detailed examinations were performed once before the first exposure, then at least weekly, in the morning or before treatment. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.

Five males and five females/group were selected:

Assessment of potential test substance related neurotoxicity was performed during the last exposure week (males on Day 27; females on PPD 4). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength.

The results were tabulated with individual and mean data. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated using a scoring system.Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5.

Body weight measurement
All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD 0, 3, 7, 10, 14, 17 and 20 and on post-partum Days (PPD) 0 (within 24 hours after parturition) and 4 (before termination). The body weight of the female animals measured on gestation Days (GD) 10 and 17 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

Food consumption measurement
Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

CLINICAL PATHOLOGY

All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy. For terminal blood sampling in all selected animals (5 males and 5 females/group), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry. For the urinalysis, the urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.

Sacrifice and pathology:

Gross necropsy was performed on all animals. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea were recorded in the females as applicable.

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 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids
Testes and epididymides were weighed individually. Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Tissue preservation and microscopic evaluation:

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.
In addition, on completion of the macroscopic examination the following tissues and
organs were retained from all animals:

Adrenal gland, Lymph node, Small intestine, Ovary Spinal cord, Aorta, Oviduct, Spleen, Brain, Pancreas Sternum with marrow, Epididymis, Pituitary, Stomach, Eye with the optic nerve, Prostate, Testis, Oesophagus, Salivary gland (including mandibular, sublingual and parotid glands), Thymus, Femur with marrow, Thyroid with parathyroid gland, Heart, Tongue, Kidney, Sciatic nerve, Trachea, Large intestine, Seminal vesicle with coagulating gland, Urinary bladder, Extraorbital lachrymal gland, Uterus, Harderian gland, Skin, subcutis with mammary gland (inguinal), Vagina, Liver.

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 male (#3011) that failed to sire and the female (#3511) that failed to deliver healthy pups.

Examination of tissues from Low and Mid dose groups were not performed as treatment-related changes were not found in the High dose group.

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.

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

Description (incidence and severity):

Occasional statistically significant differences were regarded as incidental and of no toxicological significance.

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

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Details on results:

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.

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

Conclusions:

Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male Wistar rats.

Executive summary:

A study was conducted to determine the repeated dose toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. 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. One control group and three treated groups were tested (100, 300 and 1000 mg/kg bw), each consisting of 12 males and 12 females. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, a detailed histological examination was performed on a selected list of retained organs in the control and high dose groups. Additionally, one male that failed to sire and one female that failed to deliver healthy pups in the mid dose group were also examined. No substance-related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters were observed throughout the study. No substance-related macroscopic findings were recorded in any of the dose groups at necropsy; no microscopic effects were seen at histopathology. There were no statistically significant differences among groups in the weights of organs when compared to controls. Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male Wistar rats (Weisz, 2017).

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A study was conducted to determine the repeated dose toxicity of the test substance when administered to Crl:WI Wistar rats according to OECD Guideline 422, in compliance with GLP. 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. One control group and three treated groups were tested (100, 300 and 1000 mg/kg bw), each consisting of 12 males and 12 females. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment including functional observation battery (FOB) and measurements of the landing foot splay, grip strength and motor activity were performed during the last week of the treatment. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, a detailed histological examination was performed on a selected list of retained organs in the control and high dose groups. Additionally, one male that failed to sire and one female that failed to deliver healthy pups in the mid dose group were also examined. No substance-related mortality, clinical adverse effects, or changes in neurological assessment, body weight, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters were observed throughout the study. No substance-related macroscopic findings were recorded in any of the dose groups at necropsy; no microscopic effects were seen at histopathology. There were no statistically significant differences among groups in the weights of organs when compared to controls. Under the study conditions, the NOAEL was considered to be 1000 mg/kg bw/day for the female and male Wistar rats (Weisz, 2017).

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 repeated dose toxicity according to CLP (EC 1272/2008) criteria.