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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:
29 JAN 1997 - 28 FEB 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17).The method followed that described in the OECD Guidelines for Testing of Chemicals (Adopted: 4 April 1984) No 471 "Bacterial Reverse Mutation Test".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
trans-4-pentyl-trans-4'-vinylbicyclohexyl
EC Number:
429-780-4
EC Name:
trans-4-pentyl-trans-4'-vinylbicyclohexyl
Cas Number:
129738-34-7
Molecular formula:
Hill formula: C19H34 CAS formula: C19H34
IUPAC Name:
(1r,1's,4r,4'r)-4-ethenyl-4'-pentyl-1,1'-bi(cyclohexane)
Test material form:
other: solid: wax-like

Method

Target gene:
HIS operon (S. thyphimurium), TRY operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Remarks:
his G 46, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 1537
Remarks:
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 98
Remarks:
his D 3052, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 100
Remarks:
his G 46, uvrB, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
S. typhimurium TA 102
Remarks:
his G 428, rfa + R-factor
Additional strain / cell type characteristics:
other: mutations in the histidine operon
Species / strain / cell type:
E. coli WP2
Remarks:
his C 3076, uvrB, rfa
Additional strain / cell type characteristics:
other: mutations in the tryptophan operon
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
- source of S9 : The batches of the liver homogenate fraction (S9) used in this study were prepared at the Institute of Toxicology (room U9/1092) of MERCK KGaA, Darmstadt, from October 28 to November 01, 1996. The acceptance criterium was entirely met.
- method of preparation of S9 mix : Thom (Wistar) rats (aged 6-8 weeks) were given a single intraperitoneal Injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil (MERCK, Article-No. 6175). The animals received drinking water and Altromin standard diet ad libitum. About 16 hours before sacrifice, the feed was removed. Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice-cooled sterilized beakers containing 0.15 M KCI. The livers were homogenized in a sterile glass potter homogenizer containing 3 ml of 0.15 M KCI per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen in liquid nitrogen and stored at -196°C.
- Composition and characterization of S9-Mix (quantity per mL S9-Mix):
1st series 2nd series
Liver homogenate (S9) 0.1 ml 0.2 mL
MgCI2/KCI aqueous solution (0.4 M/1.64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate 1.5 mg 1.5 mg
NADP 3.15 mg 3.15 mg
Sodium phosphate buffer (0.2 M, pH 7.4) 0.5 mL 0.5 mL
Distilled water 0.38 mL 0.18 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability). Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family.

Components added to each plate:
Bacteria suspension 0.1ml
Test material (or solvent) 0.01-0.03 ml
Sodium phosphate buffer (in studies without metabolic activation) 0.5 ml
S9-Mix (in studies with metabolic activation) 0.5 ml
Top agar solution 2.0 ml
Test concentrations with justification for top dose:
The test material concentrations were used selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1. Series: 5, 15.8, 50, 158, 500, 1580, and 5000 µg/plate
2. Series: 5, 15.8, 50, 158, and 500 µg/plate
Vehicle / solvent:
Acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) . triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
none (experiment 1), 20 min (experiment 2)
- Exposure duration/duration of treatment:
2 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored using an Artek M 880 colony counter or manually. The presence of a background lawn of non-revertant cells was checked for each plate. Tables of individual and mean values are generated by use of a validated, automated data processing program
Evaluation criteria:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.) A test material is defined as mutagenic in this assay if: - a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Statistics:
n.a.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: A weak precipitation of the test material on the agar plates was observed at concentrations > 50 µg/plate, a strong precipitation at concentrations > 500 µg/plate. Toxicity to the bacteria was not observed.


STUDY RESULTS
- Concurrent vehicle negative and positive control data
The negative control mutant frequencies were all in the regular range. Slight deviations from those values, if they occur, are accepted if they appear in only one test series and, furthermore, all other parameters of that series are in the regular range. The strain specific positive control compounds, namely daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, and cumene hydroperoxide in the absence of S9-Mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9-Mix) was functioning.


Ames test:
- Signs of toxicity : No signs of toxicity were observed (no thinning of the background lawn)
- Individual plate counts : please refer to attached pdf
- Mean number of revertant colonies per plate and standard deviation : please refer to attached pdf

Applicant's summary and conclusion

Conclusions:
According to the criteria for negative and positive results predetermined in the Study Protocol the test item was not mutagenic with and without metabolic activation (S9 mix) under the described experimental conditions.
Executive summary:

The registered substance was tested for gene mutation according to OECD Guideline 471 following GLP.

Purpose

The purpose of this assay was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations 500 µg/plate. Toxicity to the bacteria was not observed.

Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results predetermined in the Study Protocol, the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.