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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:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
29 August 2016 - 05 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Identification: (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids
Appearance: Brown solid (at 2-8°C)

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced with Aroclor 1254
Test concentrations with justification for top dose:
Experiment 1 (direct plate assay):
Preliminary test (without and with S9) TA100 and WP2uvrA: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000μg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate

Experiment 2 (pre-incubation assay):
Without and with S9-mix: 52, 164, 512, 1600 and 5000 μg/plate
Experiment 3 (pre-incubation assay):
TA1535, TA1537 and TA100, without S9-mix: 5.4, 17, 52, 164 and 512 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide. In the dose range finding test and the third mutation experiment the test item was heated up to a maximum of 62.0°C with a maximum of 45 minutes before use.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2.5 μg/plate in DMSO for TA1537 (direct plate assay)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO; 1 μg/plate for TA98 and TA100 (direct plate assay), 2.5 μg/plate for TA1535 and TA1537, 5 μg/plate for TA100 (pre-incubation assay) and 15 μg/plate for WP2uvrA.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION: Exposure duration = 48 hours; Preincubation period = 30 minutes

NUMBER OF REPLICATIONS: Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY: Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS: The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester
strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
Statistical analysis not performed

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the pre-incubation assay
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the pre-incubation assay without S9-mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the pre-incubation assay
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
Dose range finding test: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards in both tester strains and at 5000 μg/plate at the end of the incubation period in tester strain WP2uvrA only.
First mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period for all three tester strains. In addition, precipitation at the end of the incubation period was also observed at the concentration of 1600 μg/plate in tester strain TA1535 (absence of S9-mix).
Second mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period.
Third mutation experiment: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the tester strains TA100 and WP2uvrA in the direct plate assay. Precipitation of the test item on
the plates was only observed in tester strain WP2uvrA at the top dose level of 5000 μg/plate. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

HISTORICAL CONTROL DATA
The negative and strain-specific positive control values were within the testing laboratory's historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Positive historical control data:
TA1535; TA1537; TA98
S9-mix: -; +; -; +; -; +
Range: 78 - 1381; 78 - 1058; 55 - 1565; 55 – 1112; 410 - 2057; 263 - 1907
Mean: 785; 228; 653; 387; 1155; 860
SD: 167; 105; 290; 143; 370; 323
n: 1684; 1662; 1448; 1536; 1646; 1686

TA100; WP2uvrA
S9-mix: - + - +
Range: 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean: 892 1404 1263 342
SD: 178 327 461 165
n: 1650 1677 1370 1410

Negative (solvent/vehicle) historical control data:
TA1535; TA1537; TA98; TA100; WP2uvrA
S9-mix: -; +; -; +; -; +; -; +; -; +
Range: 4 - 36; 3 - 34; 3 - 25; 3 - 28; 9 - 50; 9 - 57; 63 - 153; 60 - 156; 12 - 68; 12 - 70
Mean: 14; 13; 7; 9; 17; 25; 100; 103; 26; 32
SD: 6; 5; 3; 4; 5; 7; 16; 18; 7; 8
n: 1662; 1677; 1548; 1547; 1662; 1703; 1659; 1691; 1421; 1424

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 - 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- First mutation experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence and presence of S9-mix.
- Second mutation experiment: In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. In tester strain
TA98, a slight reduction of the bacterial background lawn was observed at the top dose of 5000 μg/plate in the absence of S9-mix only and no biologically relevant decrease in the number of revertants was observed. In the tester strains TA1535, TA1537 and TA100, reductions of the bacterial background lawn and decreases in the number of revertants were observed in the absence and presence of S9-mix.
- Third mutation experiment: Cytotoxicity, as evidenced by a reduction of bacterial background lawn and/or a decrease in the number of revertants, was observed in all three tester strains.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 guideline and GLP principles, (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids was found not to be mutagenic with or without metabolic activation.
Executive summary:

An AMES test was performed according to OECD 471 guideline and GLP principles. The first experiment was a direct plate asaay and the second experiment a pre-incubation assay. All bacterial strains showed negative responses up to 5000 μg/plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. Precipitation of the test item on the plates was observed at 5000 μg/plate at the end of the incubation period. In the first experiment, cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA1537 in the absence and presence of S9-mix. In the second experiment, toxicity was observed in all Salmonella strains except for tester strain WP2uvrA. No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that (w-hydroxy) fatty acid methyl esters and (w-hydroxy) fatty acids is not mutagenic in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay with or without metabolic activation.