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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
In the study the test substance is a mixture of DL Menthol dissolved in 50% ethanol. It was obviously not the pure substance L-menthol to be registered. However, as well knows, the structure of L-menthol is very similar to D-Menthol. In addition both D & L methol share the same molecular weight, 156.27 g/mol. Existing investigations on toxicokinetics show all L-, D/L- and the unspecified menthol are well absorbed via the oral route. Additionally, for all of the isomers, elimination is rapid and mainly occurs as glucuronic acid conjugates via urine, minor amounts via faeces. Significant differences in toxicokinetic properties of menthol isomers were not found. The test substance D/L-menthol is a racemic mixture of the D- and L- isomers and contains both isomers. Data gaps for L-menthol and the unspecified isomer mixture can therefore be filled by the respective results with the racemic mixture and the doses for each isomer might be approaching to half of the total tested D/L-dose.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Special mutant strains of Salmonella typhimurium are used. Each tester strain contains a different type of mutation in the histidine operon (his-), thereby imposing a requirement for histidine in the growth medium. In addition to the histidine mutation, the tester strains contain other mutations that greatly increase their ability to detect mutagens (see Table 1). On a minimal medium containing traces of histidine the bacteria grow and divide until all histidine is consumed. Spontaneous reverse mutations to the histidine prototrophy of the wild type (his+) occurring during these few cell divisions lead to revertants. These grow in the histidine free growth medium and form visible colonies. The exposure of bacteria to various mutagens Increase the frequency of reverse mutations to the manyfold of the spontaneous value. The metabolic conversion of the test substance by mammalian liver enzymes Is examined by the comparison of the tests with and without the addition of the 59 fraction of liver homogenate.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Marenil
IUPAC Name:
Marenil
Constituent 2
Reference substance name:
89-78-1 (DL-Menthol) in 50% ethanol
IUPAC Name:
89-78-1 (DL-Menthol) in 50% ethanol
Details on test material:
test material is a mixture of DL Menthol and Ethanol

Method

Target gene:
This test uses several specially constructed histidine-requiring (his-) mutants of Salmonella typhimurium and measures his- to his+ reversion induced by chemicals which cause base changes or frameshift mutations in the genome of this organism.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate (S9) from Aroclor 1254 pretreated male rats
Test concentrations with justification for top dose:
According to an initial toxicity test HR 92/600161 was tested in concentrations of 1,5 to 150 µg per plate in the presence and of 0,5 to 50 µg in the absence of S9-mix.
Vehicle / solvent:
dimethylsulfoxide (DMSO)
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
(2-Aminoanthracene (A3880-0, Aldrich, Steinheim); 2.0 µg/plate for TA1535, TA1537, TA1538 and 0.5 µg/plate for others strains)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
H2O
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: (0.5 µg/plate TA1535 and TA100)
Negative solvent / vehicle controls:
yes
Remarks:
H2O
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: (50 µg /plate for TA1537)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Migrated to IUCLID6: (2.5 µg/plate for TA98)
Evaluation criteria:
A mutagenic activity of a substance will be suspected if :
the number of revertants on the test plates in comparison to that of spontaneous revertants on the concurrent vehicle control plates is significantly increased.
Positive results have to be reproduced and a dose related increase of the mutagenic response has to be ascertained. This might implicate the necessity of repeating the test with a narrow dosage

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results of the mutagenicity tests of the substance HR 92/600161 are summarized in Tables 1 to 4. The tables show individual plate counts and the mean number of revertant colonies per plate from two independent experiments.

The number of spontaneous revertants observed using each of the five strains was very close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979).

Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.

HR 92/600161 was tested in concentrations of 1.5 to 150 pg per plate in the presence and of 0.5 to 50 pg in the absence of S9-mix. The experiments were done with and without metabolic activation by rat liver homogenate (59). A bacteriotoxic effect was not observed both in the presence and absence of the metabolizing system.

As shown in the Tables 1 to 4, the test compound HR 92/600161 increased slightly the spontaneous mutation frequency in some of the five tester strains and at some doses tested. However, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, using a X²-test (Mohn and Ellenberger, 1977), revealed no significant difference at any one of the test points.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Menthol in Ethanol

In conclusion, these results indicate that HR 92/600161 under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA15SS, TA1537, TA1538, TA9S, and TA100 in the absence and presence c a metabolizing system.
Executive summary:

The mutagenicity of the substance HR 92/600161 (50% Menthol in Ethanol) was studied with five mutant strains of Salmonella typhimurium (TA1525, TA1537, TA1538, TA98, and TA100). The investigations were carried out using the standard plate incorporation assay with and without liver homogenate (SO) from Aroclor 1254 pretreated male rats as metabolic activation system.

HR 92/600161 was tested in concentrations of 1.5 to 150 µg per plate in the presence and of 0.5 to 50 µg in the absence of S9-mix.

Sodium azide, 2-nitrofluorene, 9-aminoacridine, and 2-aminoanthracene served as positive controls to confirm the reversion properties and the specificity of the bacterial strains as well as the efficacy of the metabolizing system.

In the concentration range investigated, HR 92/600161 did not show mutagenic activity with or without a metabolic activation system. A bacteriotoxic effect was not observed both in the presence and absence of the metabolizing system.

In conclusion, these results indicate that HR 92/600161 under the experimental conditions described, was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, and TA100 in the presence and absence of a metabolizing system.

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