Benzeneacetic acid, 2-(chloromethyl)-.alpha.-(methoxymethylene)-, methyl ester

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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 Harmonised Tripartite Guidelines S2A: Guidance on Specific Aspects of Regulatory Genotoxicity for Pharmaceuticals and S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

473-070-7

EC Name:

-

Cas Number:

117428-95-2

Molecular formula:

C12 H13 Cl O3

IUPAC Name:

methyl (2E)-2-[2-(chloromethyl)phenyl]-3-methoxyprop-2-enoate

Details on test material:

- Purity: 88.7%

Specific details on test material used for the study:

Substance name: R212143
Purity: 88.7%

Method

Target gene:

histidine synthesis

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

other: E. coli WP2P

Species / strain 3

Species / strain / cell type:

other: E. coli WP2P uvr A

Metabolic activation:

with and without

Metabolic activation system:

S-9 prepared from rat liver, phenobarbital/β-naphthoflavone induced

Test concentrations with justification for top dose:

100, 200, 500, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: Acridine Mutagen ICR 191 (TA2537 -S9), 2-Aminoanthracene (All salmonella strains and E.coli WP2P +S9), Daunomycin HCl (TA98 -S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate for test substance and positive controls. 5 plates for solvent control

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 days
- Incubation temperature: 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met: a significant, dose-related increase in the mean number of revertants is observed; a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations.

A negative result in a (valid) individual experiment is achieved when: there is no significant dose-related increase in the mean number of revertant colonies per plate observed fог the test substance; in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.

For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e., mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.

Statistics:

All derived calculations (i.e. mean colony count/plate; standard deviation, etc.) were carried out by computer. Counts from contaminated plates are not included in these calculations.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2

Remarks:

E. coli WP2P

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Test data for experiment phase 1

Test strains	±S9	Concentrations (µg per plate)
		DMSO	5000*	2500	1000	500	200	100	PC
TA1537	-S9	16 ± 5	12 ± 4	67 ± 8	57 ± 4	39 ± 6	23 ± 3	16 ± 2	32 ± 4
	+S9	14 ± 4	138 ± 57	108 ± 18	66 ± 12	51 ± 7	23 ± 1	22 ± 9	156 ± 18
TA1535	-S9	10 ± 2	17 ± 11	18 ± 4	19 ± 3	21 ± 6	14 ± 6	15 ± 2	504 ± 12
	+S9	15 ± 5	16 ± 10	22 ± 12	11 ± 4	19 ± 9	14 ± 3	26 ± 8	207 ± 33
TA98	-S9	26 ± 7	14 ± 6	21 ± 8	30 ± 3	17 ± 3	17 ± 4	27 ± 7	575 ± 24
	+S9	36 ± 7	24 ± 3	20 ± 3	27 ± 6	28 ± 6	45 ± 7	45 ± 2	1184 ± 142
TA100	-S9	102 ± 6	99 ± 17	121 ± 19	128 ± 10	106 ± 8	117 ± 11	88 ± 5	644 ± 22
	+S9	101 ± 4	56 ± 8	122 ± 38	103 ± 30	148 ± 17	133 ± 8	127 ± 8	676 ± 181
WP2P	-S9	33 ± 3	33 ± 12	28 ± 2	37 ± 7	30 ± 2	35 ± 6	36 ± 15	264 ± 8
	+S9	48 ± 6	46 ± 3	39 ± 5	47 ± 9	33 ± 7	51 ± 8	49 ± 7	199 ± 4
WP2P uvrS9	-S9	137 ± 13	108 ± 28	120 ± 24	113 ± 18	69 ± 30	120 ± 12	121 ± 10	517 ± 99
	+S9	180 ± 4	218 ± 23	144 ± 6	134 ± 23	119 ± 37	207 ± 35	229 ± 18	1009 ± 37

*Precipitate was found at 5000 µg/plate in all strains ±S9

PC = Positive Control

Table-2: Test data for experimental phase 2

Test strains	±S9	Concentrations (µg per plate)
		DMSO	5000	2500	1000	500	200	100	PC
TA1537	-S9	9 ± 3	20 ± 10	66 ± 10	32 ± 21	26 ± 13	22 ± 7	14 ± 4	45 ± 14
	+S9	12 ± 5	169 ± 49	164 ± 18	67 ± 14	40 ± 5	22 ± 6	19 ± 4	258 ± 24
TA1535	-S9	9 ± 6	16 ± 10	9 ± 6	12 ± 4	19 ± 4	21 ± 9	15 ± 4	396 ± 134
	+S9	11 ± 5	9 ± 1	10 ± 2	14 ± 8	9 ± 3	11 ± 1	7 ± 2	181 ± 17
TA98	-S9	12 ± 9	13 ± 10	9 ± 4	13 ± 7	18 ± 12	14 ± 5	5 ± 3	211 ± 81
	+S9	31 ± 5	38 ± 8	29 ± 3	39 ± 13	26 ± 5	33 ± 6	29 ± 7	1099 ± 62
TA100	-S9	104 ± 18	169 ± 31	188 ± 17	111 ± 11	138 ± 23	157 ± 25	136 ± 19	593 ± 79
	+S9	102 ± 12	48 ± 8	115 ± 6	128 ± 2	140 ± 15	125 ± 11	110 ± 7	477 ± 38
WP2P	-S9	31 ± 4	18 ± 6	32 ± 10	35 ± 5	28 ± 5	27 ± 7	39 ± 8	324 ± 16
	+S9	55 ± 5	38 ± 9	40 ± 6	47 ± 6	51 ± 5	58 ± 1	56 ± 8	280 ± 16
WP2P uvrS9	-S9	140 ± 13	113 ± 9	151 ± 26	161 ± 5	121 ± 26	125 ± 30	126 ± 18	630 ± 81
	+S9	194 ± 9	176 ± 3	219 ± 56	229 ± 25	213 ± 29	249 ± 25	249 ± 30	834 ± 62

Applicant's summary and conclusion

Conclusions:

The test substance gave a positive, i.e., mutagenic response with strain TA 1537 in both the presence and absence of S9-mix, and with strain TA 1535 in the absence of S9-mix.

Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains Salmonella typhimurium (TA 1535, TA 1537, TA98 and TA 100) and two strains of Escherichia coli (WP2P and WP2P uvrA) in the presence and absence of a rat liver - derived metabolic activation system (S9-mix). The study was conducted according to OECD guideline 471 and EU method B.13/14.

In two separate experiments, the test substance did not induce any significant, reproducible increases in the observed numbers of revenant colonies in strains TA98, ТA100, WP2P or WP2P uvrA either in the presence or absence of S9-mix, nor in strain ТА 1535 with S9-mix.

In both experiments, test substance induced significant, reproducible increases in the observed numbers of revenant colonies in strain TA1537 both in the presence and absence of S9-mix, and in strain TA1535 without S9-mix.

The sensitivity of the test system, and the metabolic activity of the S9-mix, were demonstrated by the increases in the numbers of revertant colonies induced by positive control substances.

It is concluded that, under the conditions of this assay, test substance gave a negative, i.e., non-mutagenic response in S.typhimurium strains TA98 and TA100 and E.coli strains WP2P and WP2P uvrA in both the presence and absence of S9-mix, and in strain TA1535 in the presence of S9-mix. The test substance gave a positive, i.e., mutagenic response with strain TA1537 in both the presence and absence of S9-mix, and with strain TA1535 in the absence of S9-mix.