Hexamethylene diisocyanate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: acceptable, well documented study report which meets basic scientific principles

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Reference 2

Reference Type:

publication

Title:

Lack of mutagenic activity of 1,6-hexamethylene diisocyanate

Author:

Wagner VO et al.

Year:

2000

Bibliographic source:

Toxicological Sciences 55: 376-382

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

- only 1000 immature erythrocytes per animal were scored for the incidence of micronucleated erythrocytes

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene diisocyanate

EC Number:

212-485-8

EC Name:

Hexamethylene diisocyanate

Cas Number:

822-06-0

Molecular formula:

C8H12N2O2

IUPAC Name:

1,6-diisocyanatohexane

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Indianapolis, Indiana)
- Strain: Hsd:ICR (CD-1)
- Age at study initiation: approx. 7 weeks
- Weight at study initiation: males: 25-35 g; females: 20-30 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: approx. 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

other: whole body vapour inhalation

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

The test article atmosphere will be generated into the breathing air of the test animals. The test exposure atmosphere generation system employed will be determined during pre-study trials to determine the optimal equipment and operating conditions to generate accurate and precise exposure levels. The whole-body exposure chamber will have a volume of approximately 1000 liters. The chamber will be operated at a minimum flow rate of 12 liters per minute. This will provide at least 1 air change in 5 minutes (12 air changes per hour) and a T99 equilibrium time of at most 23 minutes. This chamber size and flow rate is considered adequate to maintain the animal loading factor below 5 %. The animal exposure will begin after allowing appropriate time for chamber equilibrium. At the end of the exposure, all animals will remain in the chamber for a minimum of 23 minutes (T99 equilibrium time). During this time the chamber will be flushed with clean air at the same flow rate.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

single

Post exposure period:

24 and 48 hours

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 0.15, 0.75, 1.5 ppm
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 0.14, 0.80, 1.47 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

negative control and main groups: 10 males and 10 females per dose;
positive control group: 5 males and 5 females

Control animals:

yes, sham-exposed

Positive control(s):

cyclophosphamide monohydrate, dissolved in sterile distilled water, single i.p. injection with 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow smears; number of cells scored for micronuclei: 1000 polychromatic erythrocytes (PCEs) per animal; PCE/normochromatic erythrocytes (NCE) ratio determined for 1000 PCEs per animal.

Details of tissue and slide preparation:

At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were weIl spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Evaluation criteria:

The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5 %) in the air control; the incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the air control group (p<=0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

see "Remarks on results"

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENERAL TOXICITY:
No animals died during the course of the study. Increased activity was the only clinical sign observed during the exposures for the 0.15, 0.75, and 1.5 ppm groups. The increased activity is considered to be a transient phenomenon occurring during the early stages of exposure. Signs of toxicity observed during the post-exposure period included increased activity, slow respiration, abnormal vocalization, and labored breathing. No signs were noted on Day 2, while significant signs noted on Day 3 were slow respiration and for labored breathing in 1 to 2 animals from the 0.75 and 1.5 ppmgroups. There was no apparent test substance-related effect seen on body weight gains for the 0.1 5 ppm group when compared to the air control group. A moderate to severe effect of the test substance exposure on body weight gain was observed in the 0.75 and 1.5 ppm groups. All animals in the 2 highest exposure groups that were on study until Day 3 had lost weight compared to their pretest weight. There were no treatrnent-related macroscopic changes observed at necropsy. In conclusion, mice exposed to HDI vapors for 6 hours at 0.15 ppm had no apparent test substance effects for up to 48 hours post-exposure. Mice exposed to 0.75 and 1.5 ppm of HDI vapors for 6 hours showed considerable weight Iosses up to 24 and 48 hours post-exposure.
RATIO OF POLYCHROMATIC ERYTHROCYTES TO TOTAL ERYTHROCYTES:
Reduction of 2 to 17 % in test substance treated males at 48 hours (at 0.15 ppm 17%; at 0.75 ppm 8% and at 1.5 ppm 2%).
MICRONUCLEATED POLYCHROMATIC ERYTHROZYTES:
No significant increase was observed in male and female mice at 24, or 48 hours after exposure.

Any other information on results incl. tables

Table 1: Summary of results from the bone marrow micronucleus test with HDI

Treatment	Sex	Time (hr)	Number of mice	PCE/Total erythrocytes (Mean ± sd)	Change from control (%)	Micronucleated poly- chromatic number per 1000 PCEs (Mean ± sd)	Erythrocytes number per PCEs scored
Air
	M	24	5	0.47 ± 0.04	---	0.4 ± 0.55	2 / 5000
	F	24	5	0.45 ± 0.05	---	0.6 ± 0.89	3 / 5000
HDI
0.15 ppm	M	24	5	0.50  ± 0.11	6	0.2 ± 0.45	1 / 5000
	F	24	5	0.47 ± 0.08	4	0.4 ± 0.55	2 / 5000
0.75 ppm	M	24	5	0.50 ± 0.05	6	0.8 ± 0.84	4 / 5000
	F	24	5	0.54 ± 0.10	20	0.2 ± 0.45	1 / 5000
1.5 ppm	M	24	5	0.52 ± 0.14	11	0.4 ± 0.55	2 / 5000
	F	24	5	0.53 ± 0.09	18	0.4 ± 0.55	2 / 5000
CP
50 mg/kg	M	24	5	0.49 ± 0.12	4	32.6 ± 10.74	* 163 / 5000
	F	24	5	0.41 ± 0.06	-9	34.0 ± 5.70	* 170 / 5000
Air
	M	48	5	0.59 ± 0.12	---	0.2 ± 0.45	1 / 5000
	F	48	5	0.54 ± 0.11	---	0.4 ± 0.55	2 / 5000
HDI
0.15 ppm	M	48	5	0.49 ± 0.07	-17	0.2 ± 0.45	1 / 5000
	F	48	5	0.58 ± 0.06	7	0.4 ± 0.55	2 / 5000
0.75 ppm	M	48	5	0.54 ± 0.12	-8	0.2 ± 0.45	1 / 5000
	F	48	5	0.57 ± 0.11	6	0.2 ± 0.45	1 / 5000
1.5 ppm	M	48	5	0.58 ± 0.09	-2	0.4 ± 0.55	2 / 5000
	F	48	5	0.55 ± 0.06	2	0.2 ± 0.45	1 / 5000

* = p ≤ 0.05 (Kastenbaum-Bowman Tables)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative

Executive summary:

No significant increase in micronucleated polychromatic erythrocytes in test substance treated groups relative to the respective air control group was observed in male or female mice at 24, or 48 hours after exposure to vapour concentrations up to and including 1,47 ppm (p>0.05). The results of the assay indicate that HDI did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. HDI was concluded to be negative in the mouse micronucleus assay.