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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DIMETHYLBENZYLIDENECYCLOPENTANONE 95%
- Physical state: Solid (powder), white
- Analytical purity: 96.6%
- Lot/batch No.:COD-000462
- Storage condition of test material: Room temperature

Method

Target gene:
X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9 mix
Test concentrations with justification for top dose:
1st experiment
without S-9 mix: 0; 0.312; 0.625; 1.25; 2.5; 5.0; 10.0; 20.0 µg/mL
with S-9 mix: 0; 0.937; 1.875; 3.75; 7.5; 15.0; 30.0; 60.0 µg/mL
2nd experiment
without S-9 mix: 0; 7.5; 10.0; 12.5; 15.0; 17.5; 20.0 µg/mL
with S-9 mix: 0; 20; 40; 60; 80; 100; 120 µg/mL
Vehicle / solvent:
acetone
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without metabolic activation 300 µg ethyl methane sulfonate/ mL medium; With metabolic activation (S-9 mix) 10 µg methylcholanthrene/ mL medium
Details on test system and experimental conditions:
Exposure period
After the attachment period of 20-24 h, the medium was replaced by fresh medium. The test article, dissolved in 200 µL acetone, was added to the culture medium:
Without S-9 mix
- 20 mL Ham's F12 medium without FCS + 200 µL acetone/test substance preparation
- 18 mL Ham's F12 medium without FCS + 2 mL positive control substance
With S-9 mnix
-16 mL Ham's F12 medium without FOS + 200 µL acetone/test substance preparation
-14 mL Ham's F12 medium without FCS + 2 mL positive control substance
plus 4 mL S-9 mix ( = 2% S-9 fraction in the 1st and 2nd experiment)
Exposure lasted over a period of 4 hours both with and without S-9 mix in an incubator (5%
CO2, 37°C and >= 90% humidity).

Expression period
After the exposure period, the serum-free medium was replaced by 20 mL Ham's F12 medium with 10% FCS after having been rinsed twice with Hanks' balanced salt solution (HBSS). The following 1st passage was carried out after an incubation period of 65 – 72 hours. After an entire expression period of 6 - 8 days the cells were transferred into selection medium (2nd passage).

Selection period
For the selection of the mutants, 6 x 300 000 cells from each treatment group were seeded in six 75 cm2 flasks with about 10 mL selection medium ("TG medium") at the end of the expression period and the flasks then returned to the incubator for about 6 - 8 days. At the end of the selection period, colonies were fixed with methanol, stained with Giemsa and counted.
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S-9 mix).
- The background mutant frequencies in the negative controls should fall within the range of
0 - 15 mutants per 10e6 clonable cells (historical negative control data listed in the Appendix of the report).
- The positive controls both with and without S-9 mix must induce significantly increased mutant frequencies (historical positive control data listed in the Appendix of the report).
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently nontoxic substances are not tested at doses/concentrations higher than 5 mg/mL/ 10 mm.

Assessment criteria
A finding is assessed as positive if the following criteria are met:
- Increases of the corrected mutation frequencies both above the concurrent negative control values and the historical negative control range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.

Isolated increases of mutant frequencies above the historical negative control range (i.e. 15 mutants per 10e6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test chemical is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency in all dose groups is within the historical control range and is not significantly above the concurrent negative control.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY
Without S-9 mix, there was a decrease in the number of colonies (CE) and a reduced celI density at 20 µg/mL after an exposure period of 4 hours in the 1st experiment and a decrease in the number of colonies (CE) from about 12.5 µg/mL in the 2nd experiment onward but no reduced ceII density.
With S-9 mix, cytotoxic effects were not observed in the 1st experiment, but there was a deorease in the number of colonies (CE,) from about 60.0 µg/mL onward in the 2nd experiment. CeII density was not reduced.

CELL MORPHOLOGY
CeIl attachment was not influenced at any dose evaluated.

TREATMENT CONDITIONS
Osmolarity and pH values were not influenced by test substance treatment.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutant frequency & Cytotoxicity

1st experiment
without S-9 mix; 4-hour exposure period
Treatment (µg/mL) Metabolic activation MFuncorr (per 10e6 cells) MFcorr (per 10e6 cells) abs. cloning efficiency (%; survival) rel. cloning efficiency (%, survival) abs. cloning efficiency (%; viability) rel. cloning efficiency (%, viability)
vehicle control no 2.50 3.06 91.7 100.0 87.3 100.0
0.31 no 3.06 4.26 91.5 99.8 74.3 85.1
0.63 no 1.67 2.06 84.3 91.9 80.1 91.8
1.25 no 0.84 0.98 90.2 98.4 89.1 102.1
2.50 no 0.56 0.73 81.0 88.3 82.3 94.3
5.00 no 2.22 2.78 95.1 103.7 83.8 96.0
10.00 no 1.39 2.12 73.7 80.4 66.4 76.1
20.00 no 0.84 1.28 2.7 2.9 62.4 71.5
positive control no 59.45 79.36 95.5 104.1 75.2 86.1
withS-9 mix; 4-hour exposure period
Treatment (µg/mL) Metabolic activation MFuncorr (per 10e6 cells) MFcorr (per 10e6 cells) abs. cloning efficiency (%; survival) rel. cloning efficiency (%, survival) abs. cloning efficiency (%; viability) rel. cloning efficiency (%, viability)
vehicle control yes 0.56 0.66 81.3 100.0 84.3 100.0
0.94 yes 0.28 0.33 70.0 86.1 80.8 95.8
1.88 yes 1.39 1.93 74.7 91.9 71.8 85.2
3.75 yes 4.45 4.82 75.8 93.2 93.4 110.8
7.50 yes 0.84 0.87 86.4 106.3 85.8 101.8
15.00 yes 0.84 1.02 87.8 108.0 86.7 102.8
30.00 yes 1.12 1.26 90.7 111.6 88.9 105.5
60.00 yes 1.95 2.29 73.2 90.0 84.8 100.6
positive control yes 58.61 72.29 89.0 109.5 84.8 100.6
2nd experiment
without S-9 mix; 4-hour exposure period
Treatment (µg/mL) Metabolic activation MFuncorr (per 10e6 cells) MFcorr (per 10e6 cells) abs. cloning efficiency (%; survival) rel. cloning efficiency (%, survival) abs. cloning efficiency (%; viability) rel. cloning efficiency (%, viability)
vehicle control no 0.84 0.92 84.4 100.0 89.6 100.0
7.50 no 0.28 0.40 88.2 104.5 69.8 77.8
10.00 no 0.28 0.34 73.1 86.6 82.3 91.9
12.50 no 0.84 1.32 66.2 78.4 61.4 68.5
15.00 no 0.28 0.45 23.8 28.2 62.2 69.4
17.50 no 0.56 0.74 10.2 12.1 70.5 78.7
20.00 no 1.95 2.94 0.0 0.0 66.6 74.3
positive control no 134.73 196.55 83.2 98.6 68.6 76.6
withS-9 mix; 4-hour exposure period
Treatment (µg/mL) Metabolic activation MFuncorr (per 10e6 cells) MFcorr (per 10e6 cells) abs. cloning efficiency (%; survival) rel. cloning efficiency (%, survival) abs. cloning efficiency (%; viability) rel. cloning efficiency (%, viability)
vehicle control yes 1.67 2.07 79.7 100.0 77.2 100.0
20.00 yes 1.39 1.80 76.3 95.7 78.6 101.8
40.00 yes 1.67 2.48 68.1 85.4 66.8 86.5
60.00 yes 0.56 0.80 49.8 62.5 66.7 86.4
80.00 yes 0.56 0.63 15.9 19.9 87.2 113.0
100.00 yes 1.95 3.05 0.3 0.4 63.2 81.9
120.00 yes 1.67 2.18 0.0 0.0 69.7 90.3
positive control yes 94.17 129.43 68.9 86.4 71.5 92.6

Applicant's summary and conclusion