Reaction products of cyclohexylamine, alkan-1-amine 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:

2000

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:

Batch No.: 2008-9-16
Purity: >95.0%

Method

Target gene:

trpE ochre

Species / strain

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : purchased from a commercial source and stored at approximately -80℃.
- method of preparation of S9 mix: The S9 mix contained S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate
No evidence of toxicity was obtained following exposure to test item up to 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Suspensions of test item in water (purified in-house by reverse osmosis) containing 0.15% bacteriological agar were used in this study.

- Justification for choice of solvent/vehicle: It has been shown in a previous study conducted in this laboratory that the test item is insufficiently soluble in solvents compatible with the test system.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

In the absence of S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9 mix

Details on test system and experimental conditions:

Mutation test procedure
- 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 labelled 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).
Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained, unless otherwise justified by the Study Director.

- 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.

- Stability, homogeneity and formulation analysis
The stability of test item and the stability and homogeneity of test item in the vehicle were not determined as part of this study. Analysis of achieved concentration was not performed as part of this study.

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent vehicle controls, with some evidence of a positive dose response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fai1 to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained. Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgment.

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:

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.

Any other information on results incl. tables

 

Results of Ames test for test item using Escherichia coli

 

 Table 1– results obtained in test 1 without metabolic activation 

 

Strain	Addition	Concentration per plate	Mean revertants per plate	Standard deviation	Fold increase relative to vehicle	Individual revertant colony counts
WP2 uvrA (pKM101)	Water + 0.15% agar		196.3	4.7		200, 191, 198
	Test item	5 µg	182.3	8.1	0.9	175, 191, 181
		15 µg	149.0	5.6	0.8	150, 154, 143
		50 µg	175.3	12.3	0.9	165, 189, 172
		150 µg	207.0	9.6	1.1	218, 200, 203
		500 µg	197.0	26.5	1.0	177, 227, 187
		1500 µg	197.3	22.2	1.0	221, 177, 194
		5000 µg	176.3	15.6	0.9	160, 191, 178
WP2 uvrA (pKM101)	NQO (positive control)	2 µg	1950.7	65.6	9.9	2022, 1893, 1937

 

NQO = 4-Nitroquinoline-1-oxide

 

 

Table 2– results obtained in test 1 with metabolic activation 

 

Strain	Addition	Concentration per plate	Mean revertants per plate	Standard deviation	Fold increase relative to vehicle	Individual revertant colony counts
WP2 uvrA (pKM101)	Water + 0.15% agar		186.0	9.2		188, 194, 176
	Test item	5 µg	176.3	21.5	0.9	198, 155, 176
		15 µg	200.3	26.9	1.1	231, 181, 189
		50 µg	191.3	27.2	1.0	205, 160, 209
		150 µg	185.3	23.5	1.0	204, 159, 193
		500 µg	204.7	30.9	1.1	240, 183, 191
		1500 µg	200.3	11.0	1.1	209, 188, 204
		5000 µg	168.7	18.9	0.9	177, 147, 182
WP2 uvrA (pKM101)	AAN (positive control)	10 µg	667.7	45.2	3.6	663, 625, 715

 

AAN = 2-Aminoanthracene

 

 

 Table 3– results obtained in test 2 without metabolic activation 

 

Strain	Addition	Concentration per plate	Mean revertants per plate	Standard deviation	Fold increase relative to vehicle	Individual revertant colony counts
WP2 uvrA (pKM101)	Water + 0.15% agar		141.0	3.5		137, 143, 143
	Test item	50 µg	156.0	10.1	1.1	167, 154, 147
		150 µg	195.0	22.3	1.4	213, 170, 202
		500 µg	140.0	31.0	1.0	172, 110, 138
		1500 µg	168.3	12.4	1.2	176, 175, 154
		5000 µg	150.0	27.5	1.1	174, 120, 156
WP2 uvrA (pKM101)	NQO (positive control)	2 µg	2053.3	811.8	14.6	2890, 2001, 1269

 

NQO = 4-Nitroquinoline-1-oxide

  

 

Table 4– results obtained in test 2 with metabolic activation 

 

Strain	Addition	Concentration per plate	Mean revertants per plate	Standard deviation	Fold increase relative to vehicle	Individual revertant colony counts
WP2 uvrA (pKM101)	Water + 0.15% agar		183.7	4.7		189, 182, 180
	Test item	50 µg	170.0	36.2	0.9	136, 166, 208
		150 µg	191.7	33.5	1.0	203, 154, 218
		500 µg	181.7	27.8	1.0	213, 160, 172
		1500 µg	167.3	17.0	0.9	180, 174, 148
		5000 µg	166.7	32.3	0.9	172, 196, 132
WP2 uvrA (pKM101)	AAN (positive control)	10 µg	559.7	54.9	3.0	608, 500, 571

 

AAN = 2-Aminoanthracene

 

Applicant's summary and conclusion

Conclusions:

It is concluded that 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, 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 The test procedure was in compliance with OECD 471.

 

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.