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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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

Constituent 1
Chemical structure
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 / strainopen allclose all
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 / 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 resultsopen allclose all
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
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.