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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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Methyl octadecanoate, chloro
Molecular formula:
C17H30Cl4COOCH3, C17H29Cl5COOCH3 , and C17H28Cl6COOCH3 (primary isomers based on 35-40% Cl by weight)
IUPAC Name:
Methyl octadecanoate, chloro
Constituent 2
Reference substance name:
Methyl hexadecanoate, chloro
Molecular formula:
C15H28Cl4COOCH3 and C15H27Cl5COOCH3 (primary isomers based on 35-40% Cl by weight)
IUPAC Name:
Methyl hexadecanoate, chloro
Constituent 3
Reference substance name:
Methyl dodecanoate, chloro
Molecular formula:
C19H31Cl5COOCH3 and C19H30Cl6COOCH3 (primary isomers based on 35-40% Cl by weight)
IUPAC Name:
Methyl dodecanoate, chloro
additive 1
Chemical structure
Reference substance name:
Soybean oil, epoxidized
EC Number:
232-391-0
EC Name:
Soybean oil, epoxidized
Cas Number:
8013-07-8
Molecular formula:
C57H98O12
IUPAC Name:
Epoxidized Soybean Oil
Test material form:
liquid
Details on test material:
A complex combination of chlorinated fatty acid mtethyl esters that are predominantly C18 (~ 70 – 85%) and C16 (~ 15 - 30%) in length. The chlorination levels by weight are between 35-40%. The chlorination process is largely random, though chlorination will occur first by direct addition at unsaturated sites in the unsaturated methyl esters present in the starting material.

Method

Metabolic activation:
with and without
Metabolic activation system:
5% v/v S9 mix (initial test)
10% v/v S9 mix (confirmatory test)
Test concentrations with justification for top dose:
0.0015 to 5 µL/plate; no cytotoxicity observed
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
Used without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Used with metabolic activatiion

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
C16 and C18 chlorinated fatty acid methyl esters are concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium
Executive summary:

This study was performed to evaluate the mutagenic activity of C16 and C18 chlorinated fatty acid methyl esters using the bacterial reverse mutation test on five histidine deficient mutant tester strains ofSalmonella typhimurium(i.e., TA1537, TA1535, TA98, TA100, and TA102).

C16 and C18 chlorinated fatty acid methyl esters was tested in two independent experiments in the absence and presence of the metabolic activation. Bacterial cultures were exposed to C16 and C18 chlorinated fatty acid methyl esters at 8 concentration levels (two plates/concentration) ranging from 0.0015 to 5 µL/plate in the initial toxicity-mutation test by using the plate incorporation method. Normal bacterial background lawn (no reduction in number of revertant colonies) was observed upto the concentration of 5 µL/plate, in all the tester strains (TA1537, TA1535, TA98, TA100 and TA102) both in the absence and presence of metabolic activation system (5% v/v S9 mix).

No increase in the number of revertant colonies (no mutagenic effect) was observed in the absence and presence of the metabolic activation system (5% v/v S9 mix) in any tester strain. To confirm the negative results obtained in the initial toxicity mutation test, a confirmatory mutation test was conducted by using the plate incorporation method with an increased S9 concentration, i.e., 10% v/v S9 mix and concentration spacing modification.

Bacterial cultures were exposed at 6 concentration levels (three plates/concentration) ranging from0.15625to 5 µL/plate for tester strains TA1537, TA1535, TA98, TA100, and TA102in the absence and presence (10 % v/v S9 mix) of the metabolic activation. The revertant colonies were scored after 48 hours of incubation at 37 ± 1 °C. Minimum six analysable doses were available to evaluate the assay data during both experiments. The test material did not induce any significant increase in the number of revertants, in trials with and without S9 mix, in any tester strain. Values for the negative control were within the historical control ranges of the laboratory. Positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met.Based on the results of this study, under specified experimental conditions,

C16 and C18 chlorinated fatty acid methyl esters are concluded to be non-mutagenic in the Bacterial Reverse Mutation Test using Salmonella typhimurium.