Diethyl oxalate

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:

2010-10-12 to 2010-11-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl oxalate

EC Number:

202-464-1

EC Name:

Diethyl oxalate

Cas Number:

95-92-1

Molecular formula:

C6H10O4

IUPAC Name:

diethyl oxalate

Test material form:

other: colourless oily liquid

Details on test material:

- Name of test material: Diethyloxalate

Method

Target gene:

gene for synthesis of histidine or tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine requiring strain

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan requiring strain

Metabolic activation:

with and without

Metabolic activation system:

Delor 106 induced postmitochondrial rat liver fraction (S9)

Test concentrations with justification for top dose:

Experiment 1 and 2: 50, 150, 500, 1500, 5000 μg/plate

Vehicle / solvent:

- Vehicle used: Dimethylsulfoxide for analysis (purity>99.9%), Merck, Lot No. K40982552 019
- Justification for choice of solvent/vehicle: solubility of the test substance

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylenediamine, 9-aminoacridine hydrochloride monohydrate, 2-aminofluorene, 2-aminoanthracene, N-methyl-N-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2 series; each with and without metabolic activation, with positive control and solvent control, triplicate plating was used at each dose level

DETERMINATION OF CYTOTOXICITY
- Method: total growth

Evaluation criteria:

The main criterion for evaluation of results was the modified two-fold increase rule, which is compatible with the application of statistical methods (Dunkel V.C. et al. and Claxton L.D. et al.). Using this rule the result was positive, when a reproducible dose-effect and/or doubling of the ratio Rt/Rc was reached.

Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240
Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
Each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent; negative controls contain 0.1 mL of DMSO for injection. All the control numbers were compared with historical ranges of mutant frequencies obtained in the laboratory. The actual numbers were in ranges of the historical numbers.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Selection of doses/toxicity: As the test substance was miscible with water, a toxicity test was performed with the maximum concentration recommended in guidelines (5000 µg per plate).
The test substance was then diluted until formation of concentration series (10-5000 µg per plate), which was tested for toxicity in strain TA 100 without metabolic activation. Neither signs of toxicity nor precipitation was observed at any dose tested.

Therefore, the first mutagenicity experiment was performed with the same highest dose as used in the toxicity test. The starting dose was diluted according to the guidelines (five different analysable concentrations with approximately half log (i.e. radical 10) intervals between test points). The doses used were 50, 150, 500, 1500 and 5000 µg per plate.
As no problems occurred at evaluation with precipitation and toxicity, the same doses were used in the second mutagenicity experiments.

Any other information on results incl. tables

STUDY RESULTS

The results of experiments are summarized in the tables below. The tables contain the dose applied per plate in µg (doses were applied to plates at a volume of 0.1 mL), amount of S9 per plate in µL, numbers of revertants on single plates, average number of revertants per plate x and its standard deviation sd, and ratio of revertants at tested dose (Rt) to the number of revertants at negative control (Rc, dimethylsulfoxide).

S. typhimurium TA 100
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	128± 2	-	30	112± 9
DMSO	0	123± 3	-	30	116± 6
50	0	128± 8	1.0	30	109± 4	0.9
150	0	123±13	1.0	30	104± 8	0.9
500	0	131± 7	1.1	30	109±11	0.9
1500	0	129± 8	1.0	30	106± 1	0.9
5000	0	126±10	1.0	30	115± 7	1.0
AS/2-AF	0	513± 1	4.2	20	1289±47	11.1
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	105±10	-	30	127± 8	-
DMSO	0	114±12	-	30	130±23	-
50	0	105± 8	0.9	30	161	1.2
150	0	105± 1	0.9	30	141± 8	1.1
500	0	113± 7	1.0	30	128±13	1.0
1500	0	104± 4	0.9	30	135±14	1.0
5000	0	106± 7	0.9	30	127±15	1.0
AS/2-AF	0	440± 1	3.8	20	765±66	5.9
				experiment III with metabolic activation
spont. rev				30	136±12	-
DMSO				30	144±15	-
50				30	139± 3	1.0
150				30	132± 3	0.9
500				30	146±14	1.0
1500				30	134± 6	0.9
5000				30	132± 7	0.9
AS/2-AF				20	1525±42	10.6
S. typhimurium TA 1535
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	20± 2	-	30	16± 4	-
DMSO	0	18± 1	-	30	17± 2	-
50	0	19± 3	1.1	30	14± 2	0.8
150	0	14± 1	0.8	30	17± 2	1.0
500	0	17± 2	1.0	30	16± 2	0.9
1500	0	17± 1	1.0	30	16± 1	0.9
5000	0	15± 2	0.8	30	18± 2	1.0
AS/2-AA	0	431±18	24.4	20	226±15	13.0
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	14± 2	-	30	14± 3	-
DMSO	0	17± 4	-	30	15± 2	-
50	0	17± 2	1.0	30	14± 4	0.9
150	0	20± 1	1.2	30	21± 1	1.4
500	0	17± 1	1.0	30	16± 3	1.1
1500	0	17± 4	1.0	30	15± 2	1.0
5000	0	19± 1	1.1	30	14± 3	0.9
AS/2-AA	0	507± 5	29.8	20	130± 9	8.7
S. typhimurium TA 98
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I with metabolic activation			experiment I with metabolic activation
spont. rev	0	36± 1	-	30	33± 3	-
DMSO	0	34± 4	-	30	31± 3	-
50	0	32± 3	0.9	30	33± 4	1.1
150	0	34± 4	1.0	30	28± 4	0.9
500	0	33± 2	1.0	30	30± 4	1.0
1500	0	33± 4	1.0	30	35± 3	1.1
5000	0	33± 3	1.0	30	36± 5	1.2
NPD/2-AF	0	345± 7	10.0	20	1188±34	38.7
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	40± 4	-	30	34± 4	-
DMSO	0	34± 7	-	30	32± 3	-
50	0	37± 7	1.1	30	36± 6	1.1
150	0	33± 3	1.0	30	31± 1	1.0
500	0	30± 3	0.9	30	32± 4	1.0
1500	0	35± 2	1.0	30	34± 6	1.1
5000	0	33± 7	1.0	30	29± 4	0.9
NPD/2-AF	0	482±11	14.3	20	1807±12	57.0
S. typhimurium TA 1537
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	11± 1	-	30	12± 3	-
DMSO	0	10± 2	-	30	11± 3	-
50	0	13± 1	1.3	30	13± 3	1.2
150	0	9± 3	0.9	30	15± 3	1.4
500	0	12± 1	1.2	30	21± 2	1.9
1500	0	10± 0	1.0	30	18± 3	1.6
5000	0	12± 2	1.2	30	14± 4	1.2
9-AAc/2-AA	0	956±21	95.6	20	177± 6	16.0
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	9± 3	-	30	13± 2	-
DMSO	0	9± 4	-	30	13± 1	-
50	0	9± 2	1.0	30	13± 3	1.0
150	0	7± 4	0.8	30	17± 2	1.3
500	0	10± 3	1.1	30	14± 3	1.1
1500	0	12± 1	1.4	30	16± 2	1.3
5000	0	14± 4	1.5	30	14± 1	1.1
9-AAc/2-AA	0	1223± 3	135,8	20	96± 8	7.4
E. coli WP2 uvrA
doses	S9	mean ± sd	Rt/Rc	S9	mean ± sd	Rt/Rc
(µg/plate)	(µl)			(µl)
	experiment I without metabolic activation			experiment I with metabolic activation
spont. rev	0	26± 3	-	100	38± 6	-
DMSO	0	23± 4	-	100	34± 6	-
50	0	22± 2	0.9	100	34± 4	1.0
150	0	22± 4	1.0	100	34± 7	1.0
500	0	21± 3	0.9	100	36± 2	1.1
1500	0	20± 1	0.9	100	35± 4	1.0
5000	0	22± 5	1.0	100	35± 4	1.0
MNNG/2-AA	0	932±40	40.5	100	355±21	10.3
	experiment II without metabolic activation			experiment II with metabolic activation
spont. rev	0	36± 4	-	100	30± 8	-
DMSO	0	32± 4	-	100	32± 4	-
50	0	30± 2	0.9	100	26± 2	0.8
150	0	28± 4	0.9	100	28± 4	0.9
500	0	31± 8	1.0	100	24± 5	0.8
1500	0	25± 6	0.8	100	31± 4	1.0
5000	0	26± 6	0.8	100	31± 4	1.0
MNNG/2-AA	0	466±27	14.5	100	336± 9	10.5

Applicant's summary and conclusion

Conclusions:

The test substance was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

Executive summary:

Test substance was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The test was performed according to EU method 13114 Mutagenicity - Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was suspended in DMSO and assayed in doses of 50-5000 µg which were applied to plates in volumes of 0.1 mL. Two series of experiments were performed with each strain - without metabolic activation and with a supematant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.