Dibutyl maleate

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: A GLP study conducted according to the OECD guideline 471.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyl maleate

EC Number:

203-328-4

EC Name:

Dibutyl maleate

Cas Number:

105-76-0

Molecular formula:

C12H20O4

IUPAC Name:

dibutyl but-2-enedioate

Details on test material:

Maleic acid dibuty ester min 98% (w/w), colorless liquid, 1003 ml, homogeneous, batch No 120000134114.

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone.

Test concentrations with justification for top dose:

EXPERIMENT I
Tester strain Dose-levels (ug/plate)
TA1535 Without S9: 2500, 1250, 625, 313, 156, 78.1
TA1537 Without S9: 156, 78.1, 39.1, 19.5, 9.77
WP2uvrA With S9: 5000, 2500, 1250, 625, 313
TA1535, TA1537 With S9: 5000, 2500, 1250, 625, 313
TA98 With S9: 5000, 2500, 1250, 625, 313, 156
TA100 Without S9: 1250, 625, 313, 156, 78.1
TA100 With S9: 625, 313, 156,78.1, 39.1

EXPERIMENT II
Tester strain Dose-levels (¿g/plate)
TA1535, TA98 Without S9:2500, 1250, 625, 313, 156, 78.1
TA1535, TA1537, TA98 With S9: 5000, 2500, 1250, 625, 313
TA1537 Without S9: 156, 78.1, 39.1, 19.5, 9.77
WP2uvrA With S9: 5000, 2500, 1250, 625, 313
TA100 Without S9: 1250, 625, 313, 156, 78.1, 39.1
TA100 With S9: 625, 313, 156, 78.1, 39.1. 19.5

EXPERIMENT III
Tester strain Dose-levels (¿g/plate)
TA1535 Without S9: 2500, 1250, 625, 313, 156, 78.1
TA1535 With S9: 78.1, 39.1, 19.5, 9.77, 4.88, 2.44
TA1537 With S9: 156, 78.1, 39.1, 19.5, 9.77, 4.88
TA98 Without S9: 313, 156, 78.1, 39.1, 19.5, 9.77
TA100 Without S9: 19.5, 9.77, 4.88, 2.44, 1.22, 0.61

Vehicle / solvent:

Nutrient Broth: oxoid nutrient broth No 2 (2.5%) was used for the preparation of liquid cultures of the tester strains
Nutrietn Agar: Oxoid nutrient broth No 2 (25g) and Difco Bacto-agar (15g) wichi were used in the Petri dishes for non-selective growth of the tester strains.
Minimal agar: it was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% glucose and poured into Petri dishes.
Top agar: it was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water. Prior to use 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM) tryptophan) was added to the top agar (100 ml).

Details on test system and experimental conditions:

A preliminary toxicity test was conducted in order to select the concentrations of the test item to be used in the main test. Also a single plate was used at each test point and positive controls were not included. Three experiments were conducted with and without S9 metabolic activation for the main test using the plate incorporation method. Three replicate plates were used at each test point. The prepared plates were inverted and incubated for approximately 72 hours at 37Celsius degree. After this period of incubation, scoring was effected by counting the number of revertant colonies on each plate.

Evaluation criteria:

Toxicity was assessed o the basis of a decline in the number of spontaneous revertants, a thining of the background lawn or a microcolony formation.

Statistics:

No specified.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 4

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Any other information on results incl. tables

Experiment I: slight toxicity was observed at higher dose-levels with all tester strains with the exception of WP2uvrA, with and without S9 metabolism, and TA98 with S9 metabolism. As no relevant increases in revertat numbers were observed with any tester strains, all treatements included a pre-incubation step.

Experiment II: a less pronounced toxic effect was noted with TA1537 and WP2uvrA tester strains without S9 metabolism and with TA98 and WP2uvrA with S9 metabolism.

Experiment III: Toxicity was observed at the two highest dose-levels with all tester strains with exception of TA100 with S9 metabolism. No increases in revertant numbers were observed at any concentration, with any tester strain, with or without S9 metabolic activation.

No precipitation of the test was observed at the end of the incubation period at any concentration in any experiment.

The positive controls gave the responses as expected.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, dibutyl maleate was negative for mutagenic activity at any dose-level with any tester strain, with or without S9 metabolic activation.

Executive summary:

Dibutyl maleate was tested in three experiments for its ability to induce gene mutations in four strains of S. typhimurium and one strain of E. coli at a maximum dose-level of 5000 u/plate. The test substance does not induce two-fold increases in the number of relevant colonies, at any dose-level, in any tester strain with or without S9 metabolic activation.