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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
Study period:
From 1999-03-08 To 1999-04-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 1a: The study was performed according to OECD 406 guideline and GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Esterol C
- Analytical purity: 78.09 % (amount of C18 esters)
- Composition of test material, percentage of components: Mixture of methylesters of saturated and unsaturated C16 to C20 fatty acids, no data
- Lot/batch No.: 9906501
- Acid number: 9.30 mgKOH/g
- Expiration date of the lot/batch: December 1999
- Physical state: light yellow liquid
- Storage condition of test material: in dark and at room temperature

Method

Species / strain
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from induced Aroclor 1254 Rat liver
Test concentrations with justification for top dose:
0, 62.5, 125, 250, 500 and 1000 µg/plate, for all tested strains in the first experiment.
0, 312.5, 625, 1250, 2500 and 5000 µg/plate, for all tested strains in the second experiment.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (-S9, TA 1535 and TA 100) (1 µg/plate); 9-aminoacridine (-S9, TA 1537) (50 µg/plate); 2-nitrofluorene (-S9, TA 98) (0.5 µg/plate); Mitomycin C (-S9, TA 102) (0.5 µg/plate); 2-Anthramine (+S9, all strains) (10 µg/plate for TA102, 2 µg/plate f
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation only for the second experiment with S9 mix.
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours

DETERMINATION OF CYTOTOXICITY
- Method: other: The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn during a preliminary toxicity test.
Evaluation criteria:
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result.
Statistics:
No statistical analysis was used.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and 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 examined
Positive controls validity:
valid
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA: The number of revertants of the vehicle and positive controls was consistent with historical
data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiments without S9 mix:
Except for a slight thinning of the bacterial lawn noted in the TA strain at 1000 µg/plate, no toxicity was noted in the first experiment. In the second
experiment, a slight to marked toxicity was noted in the TA 1537 and TA 100 strains at dose-levels >= 625 µg/plate. In the TA1535 and TA 98
strains, a slight to moderate toxicity was mainly noted at dose-levels>=1250 µg/plate.
- Experiments with S9 mix:
No toxicity was noted in the first experiment in all tester strains. In the second experiment (preincubation method) a slight to moderate toxicity was induced in the TA 1535, TA 1537 and TA 100 strains mainly at dose-levels >= 1250 µg/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number of revertants per plate (first experiment) (mean of 3 plates)

TA 1535 TA 1537 TA 98 TA 100 TA 102
Conc. [unit] - MA +MA - MA + MA - MA + MA - MA + MA - MA + MA
0 9 14 7 10 17 28 79 90 154 188
62.5 9 14 8 8 12 28 79 92 175 238
125 12 13 9 8 13 27 79 100 174 228
250 11 15 5 9 19 29 75 107 176 235
500 10 14 7 9 17 24 81 91 174 202
1000 10 11 7 8 15 26 72 93 162 241
Positive control 471 332 314 110 160 937 564 1904 932 1735

MA: metabolic activation

Table 2: Number of revertants per plate (second experiment) (mean of 3 plates)

TA 1535 TA 1537 TA 98 TA 100 TA 102
Conc. [unit] - MA +MA - MA + MA - MA + MA - MA + MA - MA + MA
0 17 18 9 12 26 34 99 115 168 190
62.5 14 19 9 7 17 31 111 89 196 142
125 11 15 5 7 22 31 86 91 211 199
250 11 15 8 5 15 27 80 77 159 191
500 11 10 6 5 17 34 67 81 158 177
1000 7 10 2 3 15 32 58 65 129 146
Positive control 385 242 385 112 161 1031 405 1106 823 1646

MA: metabolic activation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Esterol C does not show any mutagenic activity in the bacterial reverse mutation test on Salmonella typhimurium strains.
Executive summary:

In a reverse gene mutation assay in bacteria (Haddouk, 1999) strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were exposed to Esterol C (batch 99.06.501) at concentration of, 0, 62.5, 125, 250, 500 and 1000 µg/plate in the presence and absence of mammalian metabolic activation. The positive controls induced the appropriate responses in the corresponding strains. No noteworthy increase in the number of revertants was induced in all tested strains with and without metabolic activation.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.