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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:
10 Dec 2013 to 13 Jan 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
EC Number:
941-628-3
Cas Number:
1263184-87-7
Molecular formula:
C12H14Cl2O2
IUPAC Name:
bis((1S,2R,6R,7S,8R)-11-(dichloromethylidene)-6-hydroxytricyclo[6.2.1.0²,⁷]undecan-3-one)
Test material form:
solid

Method

Target gene:
His-locus and Trp-locus
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Species / strain / cell type:
E. coli, other: WP2 pKM 101
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Range in plate incorporation test (experiment I) and the pre-incubation test (experiment II): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Justification for top dose: 5000 μg/plate is the maximum recommended dose according to the test guideline
Vehicle / solvent:
- Solvent used: DMSO (purity 99%)
- Justification for choice of solvent: The solvent was chosen because of its solubilisation properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
- Preincubation period: 60 min (experiment II only)
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 2 independent experiments, each using 3 replicates

DETERMINATION OF CYTOTOXICITY
- Method: a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of the historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable concentrations should be present with at least four showing no signs of toxic effects, evident as a reduction in the number of revertants below an induction factor of 0.5.

EVALUATION OF RESULTS
A test substance is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
A concentration-dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A concentration-dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium, other: TA1537, TA1535, TA100, TA98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli, other: WP2 uvrA pKM101, WP2 pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
Precipitation: The test substance precipitated in the overlay agar of the test tubes from 2500 to 5000 μg/plate with and without S9 mix in both experiments. Precipitation of the test substance in the overlay agar on the incubated agar plates was observed at 2500 and 5000 μg/plate in experiment I and II. The undissolved particles had no influence on data recording.

NUMBER OF REVERTANT COLONIES
No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

(HISTORICAL) CONTROL DATA
- The number of colonies did not quite reach the lower limit of the laboratories historical control range in the untreated and solvent control of strain WP2 uvrA pKM101 with S9 mix in experiment II. Since these deviations are rather small, they are judged to be based on biologically irrelevant fluctuations in the number of colonies and have no impact on the outcome of the study.
- Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity: Reduced background growth and toxic effects were observed at concentrations (μg/plate) shown in Table 1 and 2 in 'Any other information on results incl. tables', respectively. The pre-experiment is reported as experiment I as at least five concentrations were analysable and the criteria under "Acceptability of the assay" were met.

Any other information on results incl. tables

Table 1. Reduced background growth

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

2500, 5000

2500, 5000

/

/

TA 1537

2500, 5000

2500, 5000

/

1000 - 5000

TA 98

2500, 5000

2500, 5000

/

2500, 5000

TA 100

2500, 5000

2500, 5000

1000 - 5000

1000 - 5000

WP2 pKM101

2500, 5000

2500, 5000

2500, 5000

5000

WP2 uvrA

pKM101

 

2500, 5000

 

2500, 5000

 

/

 

/

/ = no reduction of the background growth observed

 

Table 2. Toxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5)

Strain

Experiment I

Experiment II

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

5000

/

5000

2500, 5000

TA 1537

/

5000

5000

5000

TA 98

5000

2500, 5000

5000

5000

TA 100

5000

5000

2500, 5000

1000 - 5000

WP2 pKM101

5000

5000

2500, 5000

2500, 5000

WP2 uvrA

pKM101

 

5000

 

/

 

/

 

5000

/ = no toxic effects observed

Applicant's summary and conclusion

Conclusions:
During a GLP-compliant OECD 471 mutagenicity tests and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test substance is considered to be non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

This GLP-compliant OECD 471 study was performed to investigate the potential of the test substance to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and the Escherichia coli strains WP2 uvrA pKM101 and WP2 pKM101. The assay was performed with and without Phenobarbital/ß-naphthoflavone induced rat liver S9 metabolic activation. Each concentration, including the controls, was tested in triplicate. The test substance was tested at the following concentrations: Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.

The plates incubated with the test substance showed reduced background growth at higher concentrations with all strains used in the first experiment and some of the strains in the second experiment. Cytotoxic effects, evident as a reduction in the number of revertants (below an induction factor of 0.5), were observed at high concentrations in nearly all strains used. No increase in revertant colony numbers of any of the six tester strains was observed following treatment with the test substance at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.