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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted following an O.E.C.D. Testing guideline and the GLP regulations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Testing Guideline recommended E. coli strains were not included.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid at room temperature.
Details on test material:
As per IUCLID5 Sections 1.1, 1.2. and 1.4..

Method

Target gene:
Histidine auxotrophs, hisG46, hisC3076 and hisD3052.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: Strain TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 rat liver homogenate
Test concentrations with justification for top dose:
1.6, 8, 40, 200, 1000, and 5000 ug/plate 1st mutation study. 125, 250, 500, 1000, 2000, and 5000 ug/plate 2nd mutation study. Both trials conducted with and without rat liver dervide S9 metabolic activation preparation.

Vehicle / solvent:
DMSO
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Standard Ames/Salmonella mutation assay positive controls.

Migrated to IUCLID6: 2-NF, 9-aminoacrimide, GLU , 2-anthramine
Details on test system and experimental conditions:
All the five tester strains, with the exception of strain TA 102, were originally obtained from the UK NCTC. Strain TA 102 was originally obtained from Glaxo Group Research Limited. For all assays, bacteria were cultured for 10 hours at 37°C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA1O2). Incubation was carried out in a shaking incubator. Bacteria were taken from vials of frozen cultures, which had been checked for strain characteristics of histidine dependence, rfa character and resistance to ampicillin (TA98 and TA 100) or ampicillin plus tetracycline (TA1O2). Checks were carried out according to Maron and Ames (1983) and De Serres and Shelby (1979). All experimentation commenced within 2 hours of the end of the incubation period.

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254 and obtained from Molecular Toxicology Incorporated, USA. Batches of MolTox S-9 were stored frozen at -8O C, and thawed just prior to incorporation into the top agar. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). The 10% (v/v) rat liver S-9 mix was prepeared according to Maron and Ames (1983).

2,3-Epoxypropyl neodecanoate was tested for cytotoxicity and mutation induction in the five Salmonella tester strains. Triplicate plates without and with S-9 mix were used. Negative (solvent) and positive controls were included in quintuplicate and triplicate respectively without and with S-9 mix. Theplatings were achieved by the following sequence of additions to 2.5 mL molten agar at 46°C:

0.1 mL bacterial culture
0.1 mL test article solution or control
0.5 mL 10% S-9 mix or buffer solution

followed by rapid mixing and pouring on to Minimal Davis agar plates. When set, the plates were inverted and incubated at 37°C in the dark for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted. Colonies were counted electronically using a Seescan Colony Counter (Seescan plc). The test substance was tested for mutation in two separate experiments.
Evaluation criteria:
The assay was considered valid if the following criteria were met: 1) the mean negative control counts fell within the normal laboratory historical ranges; 2) the positive control chemicals induced clear increases in revertant numbers confirming discrimination between different strains, and an active S-9 preparation.

The test article was considered to be mutagenic if: 1) the assay was valid as above; 2) Dunnett’s test gave a significant response (p < 0.01), and the data set showed a significant dose-correlation and 3) the positive responses described in 2) were reproducible.

Statistics:
Individual plate counts from each experiment was recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Dunnett’s test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
between 1000 and 5000 ug/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test substance was also cytotoxic to strain TA 102 at dose levels of 1000 - 5000 ug/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Data Tables of tester strain mean revertant counts observed with and without S-9 metabolic activation for two indepentant experiments and the Historical control background mean values are attached below.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

It was concluded that 2,3-epoxypropyl neodecanoate did induce mutation in Salmonella typhimurium strain TA100, both in the presence and in the absence of a rat liver metabolic activation system (S-9), and Salmonella typhimurium strain TA1535 only in the presence of S-9, when tested under the conditions employed by this test system, which included treatments up to 5000 ug/plate. The pattern of response in the tester strains indicated that this mutagenic activity probably occurred via a base-pair substitution mechanism.
Executive summary:

2,3 -Epoxypropyl neodecanoate was tested for the ability to induce gene-mutation in an O.E.C.D. 471 Testing Guideline (Bacterial Gene Mutation Assay) test under the GLP regulations. The experiments were conducted with and without a rat liver derive metabolic activation system (S-9 mix). It was concluded that 2,3-epoxypropyl neodecanoate did induce mutation in Salmonella typhimurium strain TA100, both in the presence and in the absence of a rat liver metabolic activation system (S-9), and Salmonella typhimurium strain TA1535 only in the presence of S-9, when tested under the conditions employed by this test system, which included treatments up to 5000 ug/plate. In tester strain TA 1535 with S-9 mix the increase in mutant frequency reached 21 -24 - fold over the control background value at 5000 ug/plate. In strain TA 100 the increase in mutant frequencyin the presence of rat liver S-9 mix at 5000 ug/plate was 4.4 - 5 - fold the concurent control background value.