Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

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

Study period:

4 august 2009 - 26 August 2009

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

Name of test material (as cited in study report): 20231250
- Physical state: white powder
- Storage condition of test material: room temperature (ca. 20°C) in the dark

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction prepared from SD derived rats, dosed with phenobarbital and 5,6-benzoflavone, purchased from a commercial source (Moltox; Lot No. 2416).

Test concentrations with justification for top dose:

Test 1 and 2: 5; 15; 50; 150; 500; 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: test item soluble in dimethyl sulphoxide (DMSO, ACS spectrophotometric grade)

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

plates without bacteria to assess the sterility of the test substance, S9 mix and PBS

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/2

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

Remarks:

Without S9 mix

Controls 2

Untreated negative controls:

no

Remarks:

plates without bacteria to assess the sterility of the test substance, S9 mix and PBS

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see Table 7.6.1/2

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in the first experiment and in the second experiment with S9 mix; preincubation in the second experiment withour S9 mix (30 min at 37°C)

DURATION
- Preincubation period: 30 min
- Exposure duration: 72 hours approximately

NUMBER OF REPLICATIONS: 3 plates/concentrations, 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: 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.

Evaluation criteria:

Acceptance criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain 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 (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.

Criteria for assessing mutagenic potential:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) 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 fail 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.

Statistics:

none

Results and discussion

Test results

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see tables 7.6.1/3 to 7.6.1/6

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Precipitate observed on all plates containing the test item at 1500 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: nodata
- Evaporation from medium: none
- Water solubility: not soluble in water but in DMSO
- Precipitation: in both experiments, precipitate observed on all plates containing the test item at 1500 and 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES: not applicable

COMPARISON WITH HISTORICAL CONTROL DATA: 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.

Any other information on results incl. tables

7.6.1/3: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the first test (direct plate incorporation method)

20231250 Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		E. coli WP2
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	26.7	2.5	13.3	2.9	39.0	1.0	161.7	6.7	170.3	5.7
5	27.7	4.7	17.3	2.5	35.0	6.1	169.7	20.4	153.3	9.8
15	30.0	10.4	19.3	2.1	34.3	2.5	159.7	19.4	167.7	13.1
50	28.0	7	16.7	2.9	45.3	4.2	153.3	18.7	177.3	5.5
150	22.0	3.5	15.0	1.7	42.0	4.4	152.0	11.1	169.3	7.6
500	26.0	3.6	21.0	1.0	47.3	4.7	146.0	7.9	160.3	10.1
1500	25.3	2.9	13.3	4.0	34.0	2.0	161.3	22.7	178.3	11.0
5000	24.0	3.6	9.7	1.5	28.7	5.5	155.3	4.5	164.7	8.1
Positive control	1063.7	79.7	913.0	138.4	256.3	114.6	961.3	79.0	2531.0	132.7

*Solvent control = negative control: 100 µL DMSO

 7.6.1/4: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the first test (direct plate incorporation method)

20231250 Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		E. coli WP2
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	27.3	0.6	28.7	3.1	54.7	6.7	197.7	11.1	196.3	3.1
5	24.0	3.5	27.3	5.8	51.7	6.1	151.3	13.0	211.3	28.5
15	23.3	2.3	21.3	4.6	50.0	6.6	158.7	11.0	180.7	23.1
50	25.0	2.6	30.3	5.8	50.7	5.0	166.7	12.7	196.3	4.9
150	21.7	2.1	34.0	4.6	55.0	4.0	175.7	6.5	203.0	15.7
500	27.0	4.6	32.7	2.1	54.3	5.1	176.3	14.4	167.0	13.5
1500	26.7	3.1	27.3	3.8	39.7	7.8	182.0	13.1	165.3	12.1
5000	22.7	2.1	25.3	1.2	46.0	2.0	156.7	10.7	159.0	10.5
Positive control	307.3	23.9	193.0	12.5	206.7	17.5	3014.7	216.5	601.3	24.8

*Solvent control = negative control: 100 µL DMSO

 

 7.6.1/5: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation in the second test (preincubation method)

20231250 Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		E. coli WP2
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	23.0	0.0	14.0	1.7	41.0	2.6	161.0	18.7	195.7	7.5
5	20.3	3.8	16.3	0.6	37.0	1.7	172.3	15.6	160.7	22.6
15	15.3	3.8	16.7	1.5	38.0	2.6	150.0	12.8	182.0	21.8
50	16.0	1.7	18.0	3.5	42.0	8.0	139.3	12.2	194.7	15.3
150	24.3	2.9	16.0	1.0	43.0	2.6	137.3	6.0	188.7	6.7
500	22.3	4.0	13.0	1.7	39.7	9.0	121.3	3.2	173.7	2.3
1500	20.7	1.5	12.7	1.5	34.7	2.5	139.7	5.5	157.3	22.1
5000	16.7	2.1	11.3	1.5	27.0	6.9	138.7	15.0	189.0	11.5
Positive control	987.0	23.4	918.3	74.7	378.3	35.1	802.0	18.0	1878.0	162.4

*Solvent control = negative control: 100 µL DMSO

 

Table 7.6.1/6: Number of revertants per plate (mean of triplicates) in the presence of metabolic activation in the second test (preincubation method)

20231250 Concentration (µg/plate)	TA 1535		TA1537		TA 98		TA 100		E. coli WP2
	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation	Mean	Standard deviation
0*	19.0	2.6	27.3	1.5	49.3	1.2	195.7	23.4	220.0	10.1
5	14.3	3.8	29.7	2.1	52.7	5.8	193.0	23.5	221.3	36.6
15	21.7	2.1	29.0	5.0	51.0	10.8	174.3	16.8	205.3	19.4
50	18.3	4.6	30.7	2.3	55.0	10.5	156.0	3.6	202.7	24.1
150	17.7	5.9	24.7	2.3	54.0	1.0	171.3	5.0	190.3	39.7
500	17.7	5.5	36.7	4.7	51.3	10.0	171.0	2.6	188.3	13.2
1500	17.3	3.1	24.0	2.6	44.7	2.3	149.7	9.9	170.0	15.7
5000	15.0	2.6	20.0	6.6	42.0	6.2	129.3	26.1	158.3	10.2
Positive control	287.3	26.1	138.0	28.6	187.7	9.9	4437.7	129.6	713.0	55.1

*Solvent control = negative control: 100 µL DMSO

Applicant's summary and conclusion

Conclusions:

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and E.coli WP2, either in the presence or in the absence of a rat liver metabolizing system.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD No.471 and EC No.B13/14 guidelines, and in compliance with the GLP, Salmonella typhimurium strains TA1535, TA1537, TA100, TA98 and E. Col WP2 were exposed to the test item diluted in DMSO at concentrations of 5; 15; 50; 150; 500; 1500 and 5000 µg/plate (triplicates) in the presence and absence of mammalian metabolic activation (fraction of S9from the liver of rats treated with phenobarbital and 5,6-benzoflavone). Both methods of direct incorporation or with a preincubation step were tested during two independent experiments. The negative control was the vehicle DMSO. Positive controls were used for each strain with and without metabolic activation and induced the appropriate responses in the corresponding strains.

No cytotoxicity was observed in any experiments. Precipitate was observed in all strains in both experiments from 1500 µg/plate. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test item at any concentration up to 5000 µg/plate in either the presence or absence of S9 mix.

The test item did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli as there was no evidence of induced mutant colonies over background.