Reaction products of cyclohexylamine and 4-alkylaniline and 1,1'-methanediylbis(4-isocyanatobenzene)

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1996-03-14 to 1996-04-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study in accordance with recognised test guideline but: Lot/batch No.: not stated Expiration date of the lot/batch: not stated NOTE: study deemed acceptable because spectral data for test item are available, covering before and after the test period - see section 1.4 Analytical Information.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: fine, cream, cohesive powder
- Storage condition of test material: ambient temperature

Test animals

Species:

rat

Strain:

other: CD strain

Details on species / strain selection:

The rat was used because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD rat was chosen because of the background data available for this strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited
- Age at study initiation: estimated to be 28 to 35 days old on arrival
- Weight at study initiation: 177-185 g (male); 140-148 g (female)
- Fasting period before study: none
- Housing: housed in stainless steel grid cages, with mesh floors and lids. The cages were suspended in a battery capable of holding up to 21 cages, above absorbent paper. The paper was changed three times per week; cages, cage-trays and water bottles were changed when necessary. Five rats of one sex were held in each cage.
- Diet (e.g. ad libitum): commercially available complete, pelleted, laboratory rodent diet was available for the rats to consume ad libitum, except overnight before blood sampling. This was an expanded autoclaved diet supplied in a discardable outer paper sack and sealed inner sterilizable light-proof polythene bag. It contained no added antibiotic or other chemotherapeutic or prophylactic agent. Weighed amounts of diet were provided at intervals during each week to each cage. At the end of each treatment week, the weight of uneaten food was recorded and the food discarded.
- Water (e.g. ad libitum): free access to tap water taken from the public supply
- Acclimation period: The animals were acclimatised for at least five days before treatment commenced. During this time, their health status was assessed by daily observation.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C (range 19-25°C)
- Humidity (%): 55% RH (range 40-70% RH)
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): a 12-hour light: 12-hour dark cycle operated

IN-LIFE DATES: From: 14 March To: 11 April 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

maize oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations of the test material were prepared for administration as a series of graded concentrations in maize oil to provide the required dosages at a constant volume-dosage of 5 mL/kg bodyweight.
AlI formulations were prepared freshly each day.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The solubility of test item was assessed in all of the solvents suitable for developing an analytical method for quality control chemistry. The test item proved to be insoluble in water, 1M HCl, 1M NaOH, methanol, acetone, dichloromethane, n-octanol, hexane, ethyl acetate, toluene, pyridine, dimethyl sulphoxide, tetrahydrofuran, acetonitrile and dimethyl formamide. Since there was no suitable solvent, homogeneity and content of test item in maize oil was assessed gravimetrically. The stability of test item could not be assessed, but in view of the insolubility in all of these vehicles, is considered extremely unlikely to be unstable under the conditions used for this study. The use of a physical method also necessitated using maize oil as a vehicle instead of the intended vehicle, 0.5% aqueous methylcellulose. Before treatment commenced, the suitability of the proposed method of formulation was determined by a trial preparation, made up as for Day 1 of treatment. Homogeneity was determined by analysis of the trial formulations for the high and low dosage concentrations immediately following their preparation. Results indicated that the homogeneity of test item dispersed in maize oil was satisfactory. In the absence of a chemical analytical method, stability of test item could not be confirmed.
In addition, duplicate samples of each formulation prepared for administration on the first day of treatment (Day 1) and on one occasion in Week 4 (Day 27) of treatment were retrospectively analysed. Results indicated that the achieved concentrations were generally satisfactory on both occasions.
- Concentration in vehicle: 4, 30, 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bodyweight

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Five

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Selection of dosages (preliminary study) Three groups of five male and five female rats received test item by oral gavage at dosages of 20, 150 or 1000 mg/kg/day for seven consecutive days. The test material was administered in 0.5% w/v aqueous methylcellulose at a volume-dosage of 10 mL/kg bodyweight. There was no death and no sign of reaction to treatment. All animals achieved anticipated bodyweight gains. The dosages chosen for the main study were 20, 150 and 1000 mg/kg/day. The latter dosage is the highest normally employed on this type of study.
- Rationale for animal assignment (if not random): On arrival, the animals were non-selectively assigned to groups and cages.
- Fasting period before blood sampling for clinical biochemistry: After four weeks of treatment (Day 29) blood samples were withdrawn from the retro-orbital sinus of each rat, following overnight food withdrawal and before dosing.

Positive control:

None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
All rats were inspected regularly for visible, or otherwise sensible, signs of ill-health or reaction to treatment. Any deviations from normal were recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition.

DETAILED CLINICAL OBSERVATIONS: Yes
Although the various examinations were not specific, they were aimed at the following features:
- A preliminary daily check for deaths or morbidity.
- At least two daily examinations for evidence of systemic toxicity or ill-health, the first immediately before dosing and the second shortly after dosing.
- An additional final check for systemic toxicity, or ill-health, on all full work days.
- A detailed weekly examination including palpation.
Any abnormality in the cage trays was noted when they were cleaned. Any animal found dead was subjected, as far as possible, to necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: each rat was weighed on the day that treatment commenced and twice weekly throughout the study period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food eaten by each cage of rats was calculated weekly by measurement of the amount of food given and that remaining in the food hoppers, together with an estimate of any food scattered.

FOOD EFFICIENCY:
Food conversion efficiency was calculated for each sex-group at weekly intervals.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water consumption was assessed visually in the course of daily observation. Quantitative measurements were not undertaken.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
After four weeks of treatment (Day 29) blood samples were withdrawn from the retro-orbital sinus ofeach rat, following overnight food withdrawal and before dosing. The rats were anaesthetized with a regulated mixture ofoxygen, nitrous oxide and Halothane during the sampling procedure.
Using EDTA anticoagulant, all samples were examined for the following characteristics:
Using a Technicon HI haematology analyser -
Packed cell volume (FCV)
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Total and differential* leucocyte count (WBC)
Platelet count (PLAT)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV).
* The equipment distinguishes neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and a small proportion of large unstained cells (LU).
Blood film - Romanowsky stain, examined by light microscopy for abnormal morphology and unusual cell types including normoblasts.

CLINICAL CHEMISTRY: Yes
Blood chemistry:
At the same time as for haematology (Day 29), a further blood sample was taken from each animal using lithium heparin as anticoagulant. After separation, the plasma was examined in respect of:
Alanine amino-transferase activity (ALT) - by the method defined by the International Federation of Clinical Chemistry, Committee on Standards, Enzyme Panel. (1978), Clin. Chem. 24: 720-721.
Aspartate amino-transferase activity (AST) - by the method defined by the International Federation of Clinical Chemistry, Committee on Standards, Enzyme Panel. (1978), Clin. Chem. 24:720-721.
Urea concentration - after Talke and Schubert (1965), Klin. Wochenschr. 43, 174.
Creatinine concentration - after Henry (1974), in "Clin. Chem. Principles and Technics", 2nd Edition Harper and Row, Hagerstown Md.
Glucose concentration (GLUC) - after Bondor and Mead (1974), Clin. Chem. 20, 586.
Total bilirubin concentration (BILT) - after Walters and Gerarde (1970), Microchem. J. 15, 231.
Total protein concentration (TP) - after Weichselbaum (1964), Am. J. Clin. Pathol. Tech. Sect. lQ, 40.
Electrophoretic protein fractions - using cellulose acetate strips, staining with Ponceau-S, and scanning with a suitable densitometer.
Sodium (NA) and potassium (K) concentrations - using the Beckman system E2A electrolyte analyser.
Chloride concentration (CL) - after Zal et al. (1956), Anal. Chern. 28, 1665.
The albumin to globulin ratio was calculated from total protein and albumin values.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Macroscopic pathology:
The procedure included a review of the history of each animal and a detailed examination of the external features and orifices, the neck and the subcutaneous structures and the cranial, thoracic, pelvic and abdominal cavities and their viscera.
External and cut surfaces of the organs and tissues were examined before or after weighing, as appropriate. Abnormalities, interactions and changes were recorded, and the required tissues preserved in fixative. Before disposing of the carcase, the tissues retained were checked against the protocol and a senior prosector reviewed the necropsy report.

Organ weights:
The organs (Adrenals, Kidneys, Liver, Testes), taken from all animals, were dissected free of adjacent fat and other contiguous tissue and the weights recorded. Organ weights relative to bodyweight gain were calculated for all animals killed after four weeks.

HISTOPATHOLOGY: Yes
Samples of Adrenals, Heart, Kidneys, Liver, Spleen and Testes were preserved in 4% neutral buffered formaldehyde, except testes which were retained in Bouin's fixative. Samples were also taken of all macroscopic abnormalities; where appropriate these samples included adjacent normal tissue.

Other examinations:

Histology and microscopic examination - the preserved tissues from all animals were dehydrated and embedded in paraffin wax, sectioned at approximately five microns thickness and stained with haematoxylin and eosin. Both auricular and ventricular sections of the heart and sections from two lobes ofthe liver were prepared. For paired organs, one section was prepared for the left and one for the right side. The sections from all animals were examined for micropathological change.

Statistics:

Inter-group differences in group mean bodyweight change, haematology and blood chemistry were evaluated by Student's 't'-test using a pooled variance. The results of this test are not presented for eosinophil, basophil, monocyte or large unstained cell counts where the data are clearly not normally distributed. For organ weights, homogeneity of variance was tested using Bartlett's test. If this was found to be statistically significant, a Fisher-Behrens test was used to perform pairwise comparisons, otherwise Dunnett's test was used. Inter-group differences in the incidence of macro- or micropathological lesions were assessed by the Fisher Exact Probability test.
Two-tailed analyses were undertaken unless otherwise indicated. Levels of statistical significance were chosen as p < 0.05 (a), p < 0.01 (b) and p < 0.001 (c) for Student's 't'-test and p < 0.05 (a) and p < 0.01 (b) for Dunnett's or Fisher-Behrens tests and the Fisher Exact Probability test. Inter-group differences that were not statistically significant (p > 0.05) are not annotated.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs of reaction to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female treated at 20 mg/kg/day was found dead on the morning of Day 14; it had shown no previous clinical sign. This death was not considered to have been associated with treatment with test item.
There was no other death.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Bodyweight gain was considered to have been unaffected by treatment with test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was considered to have been unaffected by treatment with test item.

Food efficiency:

no effects observed

Description (incidence and severity):

The food conversion efficiency was considered to have been unaffected by treatment with test item.

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology was considered to have been unaffected by treatment with test item.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Blood chemistry was considered to have been unaffected by treatment with test item.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights were considered to have been unaffected by treatment with test item.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There was no macroscopic finding which was attributed to treatment with test item.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no microscopic finding which was attributed to treatment with test item.

Effect levels

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

food efficiency

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

It is concluded that administration of test item caused no clear functional disturbance or morphological change which was toxicologically significant at dosages up to and including 1000 mg/kg/day; the test substance was accordingly not classified under EEC criteria (i.e. was not harmful by repeated or prolonged exposure).

Applicant's summary and conclusion

Conclusions:

The 'no-effect' level of administration was 1000 mg/kg/day.

Executive summary:

The systemic toxic effects of the test material during its repeated daily administration by oral gavage to rats for four weeks were assessed according to OECD 407.

Three groups of five male and five female rats received test item, at dosages of 20, 150 or 1000 mg/kg/day for four consecutive weeks. The test material was administered in maize oil at a volume-dosage of 5 mL/kg bodyweight. A similarly constituted group of rats received the vehicle alone and acted as a contemporaneous control. At the end of the treatment period, the animals were killed and subjected to detailed necropsy. Selected tissues were taken and processed for microscopic examination. One female treated at 20 mg/kg/day was found dead on the morning of Day 14; it had shown no previous clinical sign. This death was not considered to have been associated with treatment with test item. There was no sign of reaction to treatment with test item. Food consumption, bodyweight gain and food conversion efficiency were considered to have been unaffected by treatment with test item. Haematology and blood chemistry were considered to have been unaffected by treatment with test item. Organ weights were considered to have been unaffected by treatment with test item. There was no macro- or micro-pathological finding which was attributed to treatment with test item. There was no clear functional disturbance or morphological change which was toxicologically significant at dosages up to and including 1000 mg/kg/day and the test substance was therefore not classified under EEC criteria (i.e. was not harmful by repeated or prolonged exposure). The 'no-effect' level of administration was 1000 mg/kg/day.