Trimethylacetic anhydride

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:

May - June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Trimethylacetic anhydride

EC Number:

216-263-1

EC Name:

Trimethylacetic anhydride

Cas Number:

1538-75-6

Molecular formula:

C10H18O3

IUPAC Name:

2,2-dimethylpropanoyl 2,2-dimethylpropanoate

Test material form:

liquid

Method

Target gene:

histidine gene locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (Plate incorporation test): 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate. Since only moderate toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

Experiment 2 (pre-incubation test):
TA 98: 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
TA 100: 3; 10; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Remaining strains: 33; 100; 333; 1000; 2500 and 5000 μg/plate

Vehicle / solvent:

DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION:

Experiment 1: in agar (plate incorporation);
Experiment 2: preincubation;

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours

Each concentration and the controls were tested in triplicate.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate in the absence of S9 mix and at 5000 in the presence of S9 mix. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in Experiment II in the presence of S9 mix at 5000 μg/plate. The undissolved particles had no influence on the data recording.

Reduced background growth or a reduction in the number of revertants were observed at the following concentrations:
TA1535 and TA 102 - none
TA 1537 - from 1000 µg/plate on without S9 mix in Experiment I and from 2500 µg/plate without S9 mix in experiment II; none with S9 mix
TA 98 - from 1000 µg/plate on without S9 mix in Experiment I and II; none with S9 mix
TA 100 - from 333 µg/plate on wihout S9 mix in Experiment I and II; with S9 mix only at 5000 µg/plate in experiment II

Any other information on results incl. tables

Table 1: Summary of results from the salmonella typhimurium reverse mutation assay of experiment 1 (plate incorporation assay) with the test substance (mean +/- SD of revertants per plate).

Substance	Dose (µg/plate)	Without metabolic activation
		TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO		11 ± 1	11 ± 3	28 ± 8	174 ± 8	527 ± 21
Untreated		12 ± 4	11 ± 5	35 ± 4	174 ± 28	561 ± 70
Test substance	3	12 ±3	10 ± 1	27 ± 7	176 ± 14	531 ± 18
	10	12 ± 4	9 ± 3	27 ± 4	171 ± 12	564 ± 28
	33	13 ± 5	11 ± 5	30 ± 10	172 ± 15	564 ± 27
	100	11 ± 4	10 ± 5	37 ± 8	149 ± 14	537 ± 14
	333	13 ± 1	11 ± 4	23 ± 9	73 ± 9	552 ± 28
	1000	9 ± 2	9 ± 2	33 ± 15	53 ± 8	520 ± 17
	2500	14 ± 5	10 ± 1	28 ± 3	31 ± 6	526 ± 38
	5000	10 ± 1	9 ± 6	5 ± 2	6 ± 3	542 ± 17
sodium azide	10	1305 ± 124			2215 ± 128
4 -nitro-o-phenylene-diamine	10			392 ± 17
4 -nitro-o-pneylene-diamine	50		85 ± 3
methyl methane sulfonate	2.0 µl					6183 ± 412
Substance	Dose (µg/plate)	With metabolic activation
		TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO		11 ± 4	15 ± 3	45 ± 0	169 ± 3	638 ± 7
Untreated		11 ± 5	17 ± 6	40 ± 8	176 ± 30	665 ± 13
Test substance	3	10 ± 1	10 ± 4	49 ± 17	173 ± 18	732 ± 9
	10	14 ± 3	10 ± 5	37 ± 2	177 ± 9	735 ± 20
	33	14 ± 3	13 ± 4	33 ± 12	182 ± 18	768 ± 12
	100	12 ± 2	12 ± 1	41 ± 3	161 ± 23	764 ± 7
	333	14 ± 3	10 ± 3	41 ± 4	163 ± 3	750 ± 10
	1000	14 ± 5	10 ± 4	42 ± 7	187 ± 7	772 ± 7
	2500	10 ± 5	12 ± 3	29 ± 3	171 ± 13	726 ± 58
	5000	9 ± 5	15 ± 4	30 ± 6	113 ± 15	698 ± 53
2 -aminoanthracene	2.5	383 ± 34	235 ± 24	5046 ± 72	4825 ± 97
2 -aminoanthracene	10.0					1501 ± 13

Table 2: Summary of results from the salmonella typhimurium reverse mutation assay of experiment 2 (pre-incubation test) with the test substance (mean +/- SD of revertants per plate).

Substance	Dose (µg/plate)	Without metabolic activation
		TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO		10 ± 4	8 ± 2	30 ± 6	147 ± 9	500 ± 47
Untreated		11 ± 2	9 ± 2	29 ± 7	193 ± 6	457 ± 32
test substance	3				145 ± 16
	10			29 ± 3	149 ± 8
	33	9 ± 2	9 ± 2	29 ± 3	142 ± 8	479 ± 17
	100	8 ± 2	8 ± 2	30 ± 9	68 ± 13	495 ± 4
	333	9 ± 3	8 ± 3	19 ± 4	51 ± 8	473 ± 20
	1000	7 ± 2	10 ± 2	15 ± 6	34 ± 9	405 ± 9
	2500	9 ± 2	8 ± 2	1 ± 1	1 ± 1	333 ± 28
	5000	8 ± 3	1 ± 1	1 ± 1	0 ± 1	297 ± 13
Sodium azide	10	1051 ± 93			1958 ± 135
4 -nitro-o-phenylene-diamine	10			352 ± 32
4 -nitro-o-phenylene-diamine	50		85 ± 19
methyl methane sulfonate	2.0 µL					4348 ± 421
Substance	Dose (µg/plate)	With metabolic activation
		TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO		15 ± 1	16 ± 5	45 ± 6	132 ± 24	633 ± 5
Untreated		12 ± 2	18 ± 6	38 ± 1	197 ± 24	667 ± 11
Test substance	3				121 ± 11
	10			44 ± 7	145 ± 4
	33	13 ± 5	16 ± 5	37 ± 11	139 ± 14	605 ± 27
	100	12 ± 5	13 ± 5	34 ± 11	151 ± 14	659 ± 67
	333	12 ± 6	15 ± 5	36 ± 5	107 ± 8	616 ± 5
	1000	14 ± 3	16 ± 5	40 ± 16	133 ± 16	646 ± 67
	2500	12 ± 4	12 ± 3	40 ± 2	89 ± 2	583 ± 72
	5000	11 ± 5	14 ± 2	38 ± 2	36 ± 8	534 ± 49
2 -aminoanthracene	2.5	385 ± 12	178 ± 28	3323 ± 333	3923 ± 461
2 -aminoanthracene						1575 ± 177

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Trimethylessigsäureanhydrid at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Applicant's summary and conclusion

Executive summary:

This study was performed to investigate the potential of the test substance to induce gene mutations (according to OECD guideline 471) according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation in concentrations up to 5000 µg/plate. It can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.