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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:
experimental study
Adequacy of study:
key study
Study period:
30 Jan 2001 - 16 Feb 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Test material form:
liquid

Method

Target gene:
Histidine gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix
Test concentrations with justification for top dose:
- 5 to 5000 µg/plate in the presence of S9
- 1.5 to 5000 µg/plate in the absence of S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to guidelines
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: standard plate incorporation

DURATION: Exposure duration 48-72h

NUMBER OF REPLICATIONS: The experiment was performed in triplicate, and repeated in full after an interval of at least 3 days.

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn measurement and reduction in revertant colonies compared to the controls
Statistics:
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 was done, using a X2-test (Mohn and Ellenberger, 1977).

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA1537 and TA102
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA1535 and TA100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium, other: TA100 and TA102
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed

HISTORICAL CONTROL DATA:
A historical overview of the revertant frequencies of the strains used in the Freiburger Labor fur Mutagenitätsprüfung of the year 2000:
Mix: -S9 +S9
Strain: spontaneous solvent control spontaneous solvent control
TA1535: 19±5 (12/31) 19±5 (11/31) 13±5 (7/23) 14±7 (6/36)
TA1537: 10±4 (5/15) 11±4 (8/16) 15±3 (11/20) 15±3 (11/21)
TA98: 25±8 (13/41) 22±7 (15/36) 26±6 (17/36) 24±5 (14/30)
TA100: 114±19 (84/142) 105±15 (76/129) 115±18 (90/149) 108±16 (85/145)
TA102: 299±34 (225/351) 281±33 (233/332) 336±51 (261/420) 322±41 (267/388)

The historical data of positive controls of the year 2000 are given in the form: strain, mutagen (concentration of mutagen in μg/plate), mean of revertants per plate (minimum/maximum). The data of test without S9 are: TA1535, NaN3 (0.7), 631±268 (180/1065); TA1537, 9-AA (50), 259±81 (131/362); TA98, 2-NF (2.5), 361±126 (190/632); TA100, NaN3 ( 0.7), 434183 (329/665); TA102, Mitomycin C (0.15) 811±156 (577/1061). The data of the tests with lot KH3900 of S9 are: TA1535, 2-AA (0.8), 161±50 (103/264); TA1537, 2-AA (1.7), 223±66 (143/331); TA98, 2-AA (0.8), 532±159 (211/845); TA100, 2-AA (0.8), 675±216 (352/1156); TA102, 2-AA (0.8), 584±174 (383/897). Additional data of the tests with lot KH3900 of S9 are: TA98, B(a)P (5.0), 523±30 (504/558); TA100, B(a)P (5.0), 801±58 (746/862).

The number of spontaneous revertants observed using each of the five strains was close to those previously established in the laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). The results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the fall activity of the metabolizing system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-In the absence of S9-mix TETRAHYDROMYRCENOL was bacteriotoxic towards the strains TA1537 and TA102 at 500 µg/plate, towards the strains TA1535 and TA100 at 1500 µg/plate, and towards the strain TA98
at 5000 µg/plate.
-In the presence of S9-mix TETRAHYDROMYRCENOL was bacteriotoxic towards the strain TA1537 at 500 µg/plate, towards the strains TA100 and TA102 at 1500 µg/plate and towards the strain TA98 at 5000 μg/plate.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, TetrahydroMyrcenol was determined to be not mutagenic.
Executive summary:

The mutagenic activity of TetrahydroMyrcenol was evaluated in accordance with OECD TG 471 and according to GLP principles. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants and background lawn, was observed. In the absence of S9-mix test material was toxic towards the strains TA1537 and TA102 at 500µg/plate, towards the strains TA1535 and TA100 at 1500µg/plate, and towards the strain TA98 at 5000µg/plate. In the presence of S9-mix test material was bacteriotoxic towards the strain TA1537 at 500µg/plate, towards the strains TA100 and TA102 at 1500µg/plate and towards the strain TA98 at 5000 μg/plate. No precipitation was observed at any of the concentrations. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 TA102), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment.