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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:
between September 11th, 2003 to October 30th 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: Paragraph 1 of Article 57-2 of Industrial Safety and Health Law" (the Ministry of Labor Notification No. 77, September 1, 1988 and the Ministry of Labor Notification No. 67, June 2, 1997)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Pellicer
IUPAC Name:
Pellicer
Details on test material:
Chemical name: Sodium salt as a condensation product of N-lauroyl-L-glutamic acid and L-lysine (disodium or trisodium salt, partly including carboxylic acid)
Secondary name: LGM
Purity: 93.7%
Description at ordinary temperature: Solid
Stability: Stable at room temperature
Stability in solvent: Stable in water
Storage condition: At room temperature in a dark place
Lot No.: 10L-12

Method

Target gene:
Histidine operon for Salmonella
Tryptophan operon for Escherichia
Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and 5,6-benzoflavone induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Dose finding study: 5, 20, 78, 313, 1250 and 5000 µg
Main study: 313, 625, 1250, 2500 and 5000 µg
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Japanese Pharmacopoeia injection solvent
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Japanese Pharmacopoeia injection solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Sodium azide used without S9 mix
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Japanese Pharmacopoeia injection solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide
Remarks:
Positve control (AF-2) used without S9 mix
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Japanese Pharmacopoeia injection solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9AA used without S9 mix
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Japanese Pharmacopoeia injection solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
B[a]P used with S9 mix

Migrated to IUCLID6: (B[a]P)
Untreated negative controls:
yes
Remarks:
(Spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Japanese Pharmacopoeia injection solvent
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthraccne
Remarks:
2AA used with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): Not applicable.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Not applicable.

DETERMINATION OF CYTOTOXICITY
- Method: The number of reversion colonies that occurred after 48 hour-incubation of the minimal glucose agar medium at 37°C was measured. The presence or absence of antibacterial activity and precipitation of the test substance were also observed.

OTHER EXAMINATIONS:
- None
Evaluation criteria:
Acceptable standards of negative (solvent) and positive control values:
The number of reversion colonies of negative (solvent) and positive controls in each plate was compared with the background data and the standard value (mean ± 2 × SD) based on the background data. In the case where the number was found within the range of the standard value, those values outside the range were considered to be obtained by the proper procedure using an appropriate strain if the comparison with background data suggested that they resulted from an incidental factor.

Acceptable standards of test results
Test results were considered obtained by the proper procedure if they met the following conditions.
1) Sterility test demonstrated that mixed bacteria were not present.
2) Negative (solvent) control values met the acceptable standard.
3) Positive (solvent) control values met the acceptable standard, and the values were more than twice in comparison with those of negative (solvent) controls.
Statistics:
Statistical procedures were not employed in the evaluation of test results.

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:
no cytotoxicity
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:
The test substance, with and without metabolic activation, did not increase the number of the reversion colonies with dose-response in any strain and did not result in an increase of more than twice in comparison with the negative (solvent) controls. The results obtained from the dose finding study and the main study were reproducible. The sterility test demonstrated that mixed bacteria were not present in nutrient broth No. 2 used in the preculture and the maximum dose of the test substance, S9mix, 0.1 M phosphate buffer solution that were used in the main study. The numbers of the reversion colonies of the negative (solvent) and positive controls were all appropriate compared with the background data. In addition, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 2-aminoanthraccne, and benzo[a]pyrene, used as positive controls, increased the number of reversion colonies in each strain more than twice in comparison with the negative (solvent) controls. These results indicated that testing was properly conducted. Moreover, environmental factors that might affect the reliability of test results and deviation from the protocol were not found.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Please see the attachments section for the table of results

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Based on the above results, we concluded that the test substance did not have the potential for reverse mutation induction under the current test conditions. The test substance, with and without metabolic activation, did not show antibacterial activity against any strain. Also, the test substance, with and without metabolic activation, did not produce precipitation in any dose levels.
Executive summary:

The potential of reverse mutation induction of sodium salt (disodium or trisodium salt, partly including carboxylic acid) as a condensation product of N-lauroyl-L-glutamic acid and L-lysine (LGM) was investigated using Salmonella typhimurium TA100, TA1535, Escherichia coli WP2 uvrA, base pair substitution mutants, and Salmonella typhimurium TA98, TA1537, frameshift mutants. The current test consisted of the dose finding study and the main study, and these studies were conducted, according to applicable test method guidelines, in order to determine the reproducibility of the results obtained from both studies. In addition, an incubation method (37°C, 20 minutes) was utilized in both study settings. Statistical procedures were not employed in the evaluation of test results. The number of the reversion colonies of a test substance treatment was compared with that of the negative (solvent) controls. If the number of the reversion colonies of a test substance treatment increased more than twice with dose-response compared with that of negative (solvent) controls, and the reproducibility of test results was found, it was determined positive. The test results are summarized below.

1. The test substance, with and without metabolic activation, did not increase the number of the reversion colonies with dose-response in any strain and did not cause an increase more than twice compared with the negative (solvent) controls. Reproducibility was confirmed for the results obtained from the dose finding study and the main study.

2. The numbers of the reversion colonies of negative (solvent) and positive controls were appropriate in comparison with the background data. In addition, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide, sodium azide, 9-aminoacridine, 2-aminoanthraccne, and benzo[a]pyrene, used as positive controls, increased the number of reversion colonies in each strain more than twice compared with the negative (solvent) controls. These results indicate that testing was properly conducted.

3. Based on the above results, we concluded that the test substance does not have the potential for reverse mutation induction under the current test conditions.