Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 17, 2011 to June 27, 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ICH S2A Genotoxicity: Specific Aspects of Regulatory Tests, Step 5

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

EC Number:

500-740-9

EC Name:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Cas Number:

162492-07-1

Molecular formula:

C38H69N9O9

IUPAC Name:

Hexamethylene diisocyanate, oligomers, reaction products with 3-methoxypropylamine

Specific details on test material used for the study:

Batch No.: 1591ZG-075; Appearance: white powder

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Main Assay I (plate incorporation method): 5000, 1580, 500, 158, 50.0 15.8, 5.00 and 1.58 ug/plate,
Main Assay II (pre-incubation assay): 5000, 2500, 1250, 625, 313, 156 and 78.1 ug/plate.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Solutions of the test substance, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test substance. Concentrations were expressed in terms of substance as received.

The following growth media were used:
Nutrient Broth: Oxoid Nutrient Broth No 2 was prepared at a concentration of 2.5 % in distilled water and autoclaved prior to use.
This was used for the preparation of liquid cultures of the tester strains.
Nutrient Agar: Oxoid Nutrient Broth No 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water (1 L) and autoclaved.
The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
Minimal Agar: Minimal medium agar was prepared as 1.5 % Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
Top Agar: "Top Agar" (overlay agar) was prepared as 0.6 % Difco Bacto-agar + 0.5 % NaCl in distilled water and autoclaved. Prior to use 10 mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 mL.

One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study.

Bacterial strains:

TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.

Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains an uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.

Main experiments:
Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point.
In addition, plates were prepared to check the sterility of the test substance solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.

Experiment I (plate-incorporation method):
The components of the assay (the tester strain bacteria, the test substance and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation.

The overlay mixture was composed as follows: overlay agar (held at 45 °C) 2 ml, test or control substance solution 0.1 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml, bacterial suspension 0.1 ml.

Experiment II (pre-incubation method):
The components were added in turn to an empty test-tube: bacterial suspension 0.1 ml, test or control substance solution 0.05 ml, S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml.

The solutions were vortexed and incubated at 37 °C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, the scoring was effected by counting the number of revertant colonies on each plate.

Rationale for test conditions:

Solubility of the test substance was evaluated in a preliminary trial using DMSO, sterile distilled water and ethanol. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. The test substance was found to be soluble in DMSO at 100 mg/ml after 20 minute sonication. This result permitted a maximum concentration of 5000 ug/plate to be used in the Main Assay I.

Evaluation criteria:

Results shows that mean plate counts for untreated and positive control plates fell within the normal historical range. In the plate incorporation assay for TA1537, the positive control value in the absence of S9 metabolism slightly exceeded the maximum acceptable value based on the historical control data. However, the revertant number was lower than the maximum value observed in the historical control data.
The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. The study was accepted as valid.
The test substance did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Main assay II: in two or three highest dose levels

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main Assay I (plate incorporation method):

- No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism.
- Dose related precipitation of the test substance, that did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations.

Main Assay II (pre-incubation method):
- Toxicity, as indicated by thinning of the background lawn, was observed at the two or three highest dose levels with all tester strains in the absence and presence of S9 metabolism.
- Dose related precipitation of the test substance, that did not interfere with the scoring, was observed at the end of the incubation period at the three highest concentrations.
- Slight increases in revertant numbers, without any dose-effect relationship, were observed with TA1537 tester strain in the presence of S9 metabolism. These increases did not reach the two fold increases and were considered to be attributable to a chance event not related to the action of the test substance.

- The sterility of the S9 mix and of the test substance solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions.
- Marked increases in revertant numbers were obtained in these tests, following treatment with the positive control items, indicating that the assay system was functioning correctly.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, the test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay at any dose level and in any tester strain, in the absence or presence of S9 metabolism. It was concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

A study was conducted to determine the test substance ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy according to OECD Guideline 471 and EU Method B13/14, in compliance with GLP. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9-mix. The test substance was used as a solution in DMSO. In Main Assay I, using the plate incorporation method, the substance was tested at the maximum dose level of 5000 µg/plate and at seven lower dose levels spaced at approximately half-log intervals: 1580, 500, 158, 50.0 15.8, 5.00 and 1.58 µg/plate. No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. Dose-related precipitation of the test substance that did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations. As no increases in revertant numbers were observed, a pre-incubation step was included for all treatments of Main Assay II. The test substance was assayed at the maximum dose level of 5000 µg/plate and at six lower dose levels spaced by a factor of two: 2500, 1250, 625, 313, 156 and 78.1 µg/plate. Toxicity, as indicated by thinning of the background lawn, was observed at the two or three highest dose levels with all tester strains in the absence and presence of S9 metabolism. Dose related precipitation of the test substance, did not interfere with the scoring, that was observed at the end of the incubation period at the three highest concentrations. Under the study conditions, the test substance did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay at any dose level and in any tester strain, in the absence or presence of S9 metabolism. It was concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism (Ciliutti, 2011).