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

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:

weight of evidence

Study period:

From 2008-12-22 to 2009-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EC Commission Directive 2000/32/ECAnnex 4D

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

Appearance: White powder
Storage conditions: Refrigerated in the dark
Batch number: 2008-9-3
Expiry date: 30 September 2009
Purity: >95.0%

Method

Target gene:

Trypophan dependancy

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Test 1: 5, 15, 50, 150, 500, 1500, 5000 µg per plate
Test 2: 50, 150, 500, 1500, 5000 µg per plate

Vehicle / solvent:

It has been shown in a previous study conducted in this laboratory that test item is insufficiently soluble in solvents compatible with the test system (Life Sciences Research report no. 91/NNG003/0816). Suspensions of test item in water (purified in house by reverse osmosis) containing 0.15% bacteriological agar were, therefore, used in this study.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

aqueous 0.15% agr solution

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

First test
Aliquots of 0.1 mL of the test substance suspensions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. The negative control was the chosen vehicle, aqueous 0.15% agar solution. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labeled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 hours. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).


Second test
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 minutes with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.

Positive controls:
In the absence of S9 mix
Identity: 4-Nitroquinoline-1-oxide
CAS No.: 56-57-5
Solvent: DMSO (A.C.S. spectrophotometric grade)
Concentration: 2 μg/plate

In the presence of S9 mix
Identity: 2-Aminoanthracene
CAS No.: 613-13-8
Solvent: DMSO (A.C.S. spectrophotometric grade)
Concentration: 10 μg/plate

Evaluation criteria:

For a test to be considered valid, the mean of the vehicle control revertant colony numbers should lie within or close to the 99% confidence limits of the current historical control range of the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 1E+09/mL.

Results and discussion

Test results

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results obtained with test item and positive control compounds are presented in Table 1 (first test) and in Table 2 (second test).
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation.
The total colony counts on nutrient agar plates (see Table 3) confirmed the viability and high cell density of the cultures of the individual organisms.
The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory (Appendix 1). Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test
No evidence of toxicity was obtained following exposure to test item. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test
No evidence of toxicity was obtained following exposure to test item.
No substantial increases in revertant colony numbers over control counts were obtained following exposure to test item at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:

The test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Executive summary:

In this in vitro assessment of the mutagenic potential of test item in complied with OECD 471, a tryptophan-dependent mutant of Escherichia coli, strain WP2 uvrA (pKM101), was exposed to test item suspended in aqueous 0.15% agar solution, which was also used as a negative control. Two independent mutation tests were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of test item up to 5000 μg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. No signs of toxicity were observed towards the tester strain in either mutation test following exposure to test item. No evidence of mutagenic activity was seen at any concentration of test item in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. It is concluded that test item showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.