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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:
experimental study
Adequacy of study:
key study
Study period:
From 27 may 2003 to 27 august 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: very viscous colorless gel
Details on test material:
- Name of test material (as cited in study report): Isobornylcyclohexanol
- Analytical purity: 100%
See confidential details on test material for more information

Method

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:
S9 mix from rat liver cells
Test concentrations with justification for top dose:
Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.
Experiments without S9 mix:
The selected treatment-levels were:
• 31.3, 62.5, 125, 250 and 500 μg/plate, for the TA 102 in both experiments,
• 7.81, 15.63, 31.3, 62.5 and 125 μg/plate for the TA 100, TA 98 and TA 1535 strains in both experiments and for the TA 1537 strain in the first experiment,
• 1.95, 3.9, 7.81, 15.63 and 31.25 μg/plate for the TA 1537 strain in the second experiment.
Experiments with S9 mix:
The selected treatment-levels were:
• 62.5, 125, 250, 500 and 1000 μg/plate, for the TA 102 in both experiments,
• 31.3, 62.5, 125, 250 and 500 μg/plate, for the TA 1535, TA 1537 and TA 100 strains in the first experiment and for the TA 98 strain in both experiments,
• 15.63, 31.3, 62.5, 125 and 250 μg/plate in the second experiment with the TA 1535, TA 1537 and TA 100 strains.
Vehicle / solvent:
Vehicle used: The vehicle was dimethylsulfoxide (DMSO), batch No. K30379650 214 (Merck Eurolab, Fontenay-Sous-Bois, France).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see details on test system and conditions
Details on test system and experimental conditions:
Positive controls:
Without S9 mix
. sodium azide (NAN3) 1 µg/plate for TA 1535 - TA 100
. 9-Aminoacridine (9AA) 50 µg/plate for TA 1537
. 2-Nitrofluorene (2NF) 0.5 µg/plate for TA 98
. Mitomycin C (MMC) 0.5 µg/plate for TA 102

With S9 mix
. 2-Anthramine (2AM) 2 µg/plate for TA 1535 - TA 1537 - TA 98 - TA 100 and 10 µg/plate for TA 102

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation method.
Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

DURATION
- Preincubation period: 60 min at 37°C
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): the number of revertants is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertants per plates are counted
Evaluation criteria:
Acceptance criteria
This study is considered valid if the following criteria are fully met:
- the number of revertants in the vehicle controls is consistent with the historical data of the testing facility,
- the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) 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. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
no data

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test:
The test item was freely soluble in the vehicle (DMSO) at 50 mg/mL.
Consequently, with a treatment volume of 100 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.
A moderate to marked emulsion was observed in the Petri plates when scoring the revertants at all dose-levels ≥ 1000 μg/plate.
Without S9 mix, moderate to strong toxicity was induced at dose-levels ≥ 100 μg/plate (TA 98 and TA 100 strains) or ≥ 500 μg/plate (TA 102 strain).
With S9 mix, moderate to strong toxicity was induced at dose-levels ≥ 500 μg/plate (TA 98 and TA 100 strains) or ≥ 1000 μg/plate (TA 102 strain).

Any other information on results incl. tables

see details on results in attached document

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative with and without metabolic activation

Under our experimental conditions, the test item Isobornylcyclohexanol (Rhodiantal IBCH) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

This study was performed to investigate the potential of the test item, Isobornylcyclohexanol (Rhodiantal IBCH), to induce reverse mutation in Salmonella typhimurium. The study was performed according to OECD guideline no. 471 and EC guideline n° B13/14 and in compliance with the Principles of Good Laboratory Practice. 

A preliminary toxicity test was performed to define the dose-levels of Isobornylcyclohexanol (Rhodiantal IBCH) to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

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. The test item Isobornylcyclohexanol was dissolved in dimethylsulfoxide (DMSO).

Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were:

• 31.3, 62.5, 125, 250 and 500μg/plate, for the TA 102 in both experiments,

• 7.81, 15.63, 31.3, 62.5 and 125μg/plate for the TA 100, TA 98 and TA 1535 strains in both experiments and for the TA 1537 strain in the first experiment,

• 1.95, 3.9, 7.81, 15.63 and 31.25μg/plate for the TA 1537 strain in the second experiment.

 

Experiments with S9 mix:

The selected treatment-levels were:

• 62.5, 125, 250, 500 and 1000μg/plate, for the TA 102 in both experiments,

• 31.3, 62.5, 125, 250 and 500μg/plate, for the TA 1535, TA 1537 and TA 100 strains in the first experiment and for the TA 98 strain in both experiments,

• 15.63, 31.3, 62.5, 125 and 250μg/plate in the second experiment with the TA 1535, TA 1537 and TA 100 strains.

 

Without S9 mix, moderate to marked toxicity was noted in the TA 1537 and TA 100 strains at dose-levels ≥ 31.3μg/plate, in the TA 98 and TA 1535 strains at dose-levels ≥ 125μg/plate and in the TA 102 strain at 500μg/plate.

With S9 mix, moderate to marked toxicity was noted in the TA 1535 and TA 100 strains at dose-levels ≥ 125μg/plate, in the TA 98 and TA 1537 strains at dose-levels ≥ 250μg/plate and in the TA 102 strain at dose-levels ≥ 500μg/plate.

The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the five strains.

Under our experimental conditions, the test item Isobornylcyclohexanol (Rhodiantal IBCH) did not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

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