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Toxicological information

Genetic toxicity: in vitro

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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:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted May 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: 8 g Difco nutrient broth + 5 g NaCI/liter
- Properly maintained: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: 8 g Difco nutrient broth + 5 g NaCI/liter
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 rat liver induced S9 mix
Test concentrations with justification for top dose:
20µg, 100µg, 500µg, 2500µg, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: low water solubility of test substance, DMSO had been demonstrated to be suitable in bacterial lreverse mutation tests and historical control data are available
Controlsopen allclose all
Untreated negative controls:
other: sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
with S9
Untreated negative controls:
other: sterility contol
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for TA 1535, TA 100; 4-nitro-o-phenylendiamine (NOPD) for TA 98; 9-aminoacridine (AAC) for TA 1537; 4-nitroquinoline-N-oxide (4-NQO) for E . coli WP2 uvrA
Remarks:
without S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; with and without preincubation

DURATION
- Preincubation period: 20min
- Exposure duration: 48h,72h

SELECTION AGENT (mutation assays): lack of tryptophane in soft agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn (=reduced his- or trp- background
growth ); reduction in the titer

Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met :
• A dose-related and reproducible increase in the number of revertant colonies, i .e . about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if :
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other .

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:
cytotoxicity
Remarks:
slight at >= 2500µg/ml (standard) or >= 500 - 2500µg/ml (preincubation)
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system.
Cytotoxicity was observed in Salmonella strains in the preincubation assay at and above 500 µg/plate - 2500 µg/plate depending on strain.

Applicant's summary and conclusion

Conclusions:
Thus, under the experimental conditions chosen here, it is concluded that the test substance is not a mutagenic agent in a bacterial reverse mutation test.
Executive summary:

In a gene mutation assay in bacteria (Ames) according to OECD471 and GLP (2002), 1,4 -butanediylbis[oxy(2 -hydroxy-3,1 -propanediyl)] diacrylate did not lead to an increase in the number of his + or trp+ revertants in both, the standard plate test and in the preincubation test either without S-9 mix or after the addition of a metabolizing system. Concentrations up to 5000µg/plate were tested in Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli 1412 uvrA. Cytotoxicity was observed in Salmonella strains in the preincubation assay at and above 500µg/plate - 2500µg/plate depending on strain. S9 fraction was prepared from aroclor 1254 induced rat liver.