Hexamethylene diisocyanate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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

Principles of method if other than guideline:

METHOD: The test system was exposed to the test substance via the desiccator methodology, a modification of the plate incorporation methodology originally described by Ames et al., Mutat. Res. 31, 347-364 (1975) and updated by Maron and Ames, Mutat. Res. 113, 173-215 (1983). The desiccator methodology has been shown to be an effective method for detecting the genotoxic activity of volatile and gaseous test substances (Wagner et al., Environ. Mol. Mutagen. 19, 68 (1992)). Deviation from the original study protocol: The independent repeat assay was not performed because technical difficulties in dosing the test article using the desiccator methodology make further testing unwarranted. The test article exposure was reduced to approx. 8 hours. After this exposure period, the plates were removed from the desiccator and incubated with the lids replaced such that the total incubation time was approx. 48 to 72 hours. No strains with an AT base pair at the primary reversion site (E. coli WP2 strains or S. typhimurium TA 102) were used to detect cross-linking mutagens.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation 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

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male rat liver S9 mix

Test concentrations with justification for top dose:

6, 12, 20, 25, 50 and 150 µL per desiccator

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Migrated to IUCLID6: 2-aminoanthracene, sodium azide, 9-aminoacridine

Details on test system and experimental conditions:

see "Principles of method"

Evaluation criteria:

For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test substance. Data sets for strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean untreated control value. Data sets for strains TA98, TAlOO and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the
mean untreated control value.

The following criteria must be met for the mutagenicity assay to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene. Cultures of tester strains TA98 and TAI00 must demonstrate the presence of the pK.MI0l plasmid R-factor. All WP2 uvrA cultures must demonstrate the deletion in the uvrA gene. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the untreated controls as follows (inclusive): TA98, 10 - 50; TAI00, 80 - 240; TA1535, 5 - 45; TA1537, 3 - 21; WP2 uvrA, 10 - 60. To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.3xl09 cellsjmL. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the mean value of the respective untreated control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A >50 % reduction in the mean number of revertants per plate as compared to the mean untreated control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitate was observed but toxicity generally observed at >= 6µL per desiccator, with non-uniform toxicity over at least 25% of the surface of each affected plate. The non-uniform toxicity did not appear to be a function of the location or orientation of the plates in the desiccators. The non-uniform toxicity profile appears to be unique to HDI. In addition, no mutagenic activity was observed in locations on the plates where the background lawns were nontoxic (normal).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of results from the Salmonella mutagenicity assay with HDI (average revertants per plate ± standard deviation)

Dose (µL per desiccator)	Without metabolic activation
	TA 98	TA 100	TA 1535	TA 1537
Untreated	15 ± 2	185 ± 9	17 ± 5	5 ± 3
6	3 ± 1	41 ± 9	3 ± 2	2 ± 2
12	6 ± 7	93 ± 16	3 ± 1	2 ± 2
20	5 ± 4	15 ± 19	2 ± 3	2 ± 2
25	0 ± 1	23 ± 8	2 ± 2	1 ± 1
50	2 ± 2	27 ± 19	2 ± 1	1 ± 1
150	1 ± 2	34 ± 32	3 ± 5	2 ± 1
Positive control	343 ± 59	667 ± 34	599 ± 7	401 ± 195
Dose (µL per desiccator)	With metabolic activation (liver S9 mix)
	TA 98	TA 100	TA 1535	TA 1537
Untreated	21 ± 3	184 ± 5	16 ± 1	6 ± 4
6	14 ± 6	92 ± 29	5 ± 1	2 ± 2
12	13 ± 8	129 ± 19	13 ± 1	4 ± 2
20	13 ± 9	71 ± 17	3 ± 3	3 ± 3
25	13 ± 9	95 ± 66	9 ± 5	1 ± 2
50	10 ± 7	120 ± 9	8 ± 2	2 ± 1
150	9 ± 2	126 ± 11	8 ± 3	4 ± 3
Positive control	940 ± 404	826 ± 157	77 ± 11	94 ± 21

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

The results of the Bacterial Reverse Mutation Assay using vapour-phase exposure for 1,6 -hexamethylene diisocyanate indicate that 1,6-hexamethylene diisocyanate does not appear to induce any mutagenic activity with any of the tester strains in the presence and absence of a metabolic activation system.