Diethyl maleate

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:

8 April 2004 to 17 January 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diethyl maleate

EC Number:

205-451-9

EC Name:

Diethyl maleate

Cas Number:

141-05-9

Molecular formula:

C8H12O4

IUPAC Name:

diethyl (Z)-but-2-enedioate

Details on test material:

Name: "DIETHYLMALEINATE".
Chemical name: Maleic acid diethyl ester.
Trade name: DeM.
Molecular formula: C8H12O4.
CAS No.: 141-05-9.
Batch No.: ETLM 201.
Appearance: Colourless liquid.
Solubilities: Poorly soluble in water, soluble in organic solvents.
Melting point: - 11 °C.
Boiling point: 226 °C.
Conditions of storage: In the dark, may be used under light.
Stability at conditions of storage: Stable.
Date of expiry: 30 June 2005.

Method

Target gene:

his- (s. typhimurium)

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA97a

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

5000, 1667, 556, 185 and 62 µg/plate

Vehicle / solvent:

DMSO

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine; t-Butyl-hydroperoxide; 2- Aminoanthracene; 1,8-Dihydroxy-anthraquinone; 7,12-Dimethylbenz[a]anthracene; 2-Nitrofluorene; Sodium acide

Details on test system and experimental conditions:

Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535, obtained from Prof. Bruce N. Ames, Berkeley, California, were used.
The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000. The bacteria were stored frozen since that time.

The test substance was tested without as well as with an external metabolising system (S9 mix). The results were verified by a second, independent experiment.
For metabolic activation (S9-Mix) the microsomal fraction of homogenised livers of rats treated once with 500 mg/kg of Aroclor 1254 was used. The preparation was done in March 2004. Four days after treatment, the feed was withdrawn for one night. The livers of the animals were removed and homogenised in cold 0.15 mol/l KCl. Three mL of homogenate were obtained per gram of liver. Then the homogenate was centrifuged for 10 minutes at 9000 x g. The supernatant contained the microsomes. Small portions of the microsomes were stored in liquid nitrogen. Immediately before use they were thawed and mixed with the cofactor solution. The metabolic activity of the microsomes was verified by the positive control substances of each study.

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 108 cells per mL.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535 with the exception of the positive controls, were counted visually by marking the colonies with a felt tipped pen. The other plates were photographed with a video camera and the picture files were scanned for colonies by a computer program.

Evaluation criteria:

The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
•For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
•For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1 2/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.

Additionally to the counting of colonies the bacterial background of the plates was inspected visually. The following signs of toxicity, if present, were recorded:
• A reduced bacterial background lawn (mottled instead of homogeneous).
• Microcolonies of bacteria instead of a homogeneous background lawn.
• No background lawn.
• Clearly reduced numbers of revertant colonies.

Statistics:

Means and standard deviation were calculated for the number of mutants in every concentration group.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium, other: TA97a

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

Species / strain:

S. typhimurium TA 102

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

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 4

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 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test substance was seen in any of the concentration groups.

Applicant's summary and conclusion

Conclusions:

negative

According to these results, "DIETHYLMALEINATE" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to 5000 μg/plate, which is the limit concentration for this kind of test.

Executive summary:

Method "DIETHYLMALEINATE" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part B.13/14. The test substance, dissolved in DMSO, was tested at concentrations ranging from 62 μg to 5000 μg per plate according to the "direct plate incorporation method" without external metabolisation as well as with an external metabolising system (S9-mix). As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 were used. Negative and positive controls were included. An independent repetition of the experiment was performed. Results Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system. Test substance: Toxicity: No toxicity of the test substance to the bacteria was observed up to 5000 μg per plate. Solubility: No precipitation of the test substance was seen in any of the concentration groups. Mutagenicity: In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results. Conclusion According to the results obtained in this study, "DIETHYLMALEINATE" is non-mutagenic in the Ames test with the strains TA97a, TA98, TA100, TA102 and TA1535 up to 5000 μg/plate, which is the limit concentration for this kind of test.