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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:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 21, 1997
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
adopted August 1998
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted May 30, 2008
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): Laromer PO 33 F
- Physical state: Liquid yellowish
- Analytical purity: The test item is a complex mixture of isomers and homologues components, so no purity can be stated. At least 88.8% of the mixture were identified as reaction products of TMP with varying amounts of acrylic acid, EO and PO (see analytical report, BASF study code 11L00272); dose calculation not adjusted to purity
- Purity test date: 02.01.2012
- Lot/batch No.: 110007P040
- Expiration date of the lot/batch: 07 July 2012
- Stability under test conditions: Not indicated by the sponsor
- Storage condition of test material: Room temperature, avoid temperature above 25°C

Method

Target gene:
HPRT
Species / strain
Species / strain:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell lines (if applicable):
- Type and identity of media: MEM containing Hank's salt supplemented with 10% FBS, 5µg/mL neomycin, 1% amphotericin B (Serum free medium was used during the 4h treatment), for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
4h without S9: 0, 0.3, 0.6, 1.3, 2.5µg/mL (additional concentrations not chosen for analysis due to cytotoxicity: 3.8, 5.0 µg/ml)
4h with S9: 40, 80, 160, 240, 300µg/mL
24h without S9: 0.32, 0.63, 1.3, 1.9, 2.5µg/ml
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relative non-toxicity to the cell cultures
Controlsopen allclose all
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with S9
Details on test system and conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4h and 24h
- Expression time (cells in growth medium): 7days
- Selection time (if incubation with a selection agent): 8days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days plus exposure duration

SELECTION AGENT (mutation assays): 6-TG

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method:cloning efficiency
Evaluation criteria:
Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 106 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.

Evaluation of Results
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory's historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at 2.5µg/ml (without S9), 300µg/ml (with S9)
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
COMPARISON WITH HISTORICAL CONTROL DATA:
all controls and all treated groups were within the historical controls

Any other information on results incl. tables

        relative relative relative mutant   relative relative relative mutant  
  conc. PS S9 cloning cell cloning colonies/ induction cloning cell cloning colonies/ induction
  1g/mL   mix efficiency I density efficiency II 106 cells factor efficiency I density efficiency II 106 cells factor
        % % %     % % %    
Column 1 2 3 4 5 6 7 8 9 10 11 12 13
Experiment I / 4 h treatment       culture I culture II
Solvent control with DMSO     - 100,0 100,0 100,0 18,9 1,0 100,0 100,0 100,0 5,8 1,0
Positive control (EMS) 150,0   - 92,6 140,2 98,2 75,7 4,0 70,7 93,5 91,3 165,1 28,3
Test item 0,3   - 97,8 118,2 97,0 9,0 0,5 94,2 136,9 102,6 12,6 2,2
Test item 0,6   - 98,0 122,2 87,8 16,7 0,9 88,9 160,7 104,8 15,1 2,6
Test item 1,3   - 75,5 116,4 84,0 24,2 1,3 75,6 119,4 108,3 30,3 5,2
Test item 2,5   - 12,7 101,2 84,4 6,5 0,3 33,1 97,1 93,9 17,3 3,0
Test item 3,8   - 0,0 culture was not continued# 24,5 3,5 culture was not continued#
Test item 5,0   - 0,0 culture was not continued# 21,4 culture was not c ontinued#
Solvent control with DMSO     + 100,0 100,0 100,0 7,5 1,0 100,0 100,0 100,0 9,2 1,0
Positive control (DMBA) 1,1   + 75,4 122,7 89,3 485,8 64,8 74,9 102,6 70,6 630,6 68,5
Test item 20,0   + 97,0 culture was not continued## 96,2 culture was not continued##
Test item 40,0   + 95,7 100,5 93,9 15,1 2,0 97,5 104,6 82,3 5,0 0,5
Test item 80,0   + 94,7 80 .3 98,4 7,9 1,1 95,6 116,6 78,3 21,5 2,3
Test item 160,0   + 92,6 86 .8 105,1 8,1 1,1 94,6 91,4 87,4 13,3 1,4
Test item 240,0 PS + 24,0 125,0 93,7 20,8 2,8 92,3 101,1 78,3 10,0 1,1
Test item 300,0 PS + 0,0 30 .3 97,1 7,9 1,1 84,3 90,4 76,1 4,0 0,4
Experiment II / 24 h treatment       culture I culture II
Solvent control with DMSO     - 100,0 100,0 100,0 21,3 1,0 100,0 100,0 100,0 3,6 1,0
Positive control (EMS) 150,000   - 119,6 80 .3 84,1 347,6 16,4 108,6 102,5 82,3 308,6 85,1
Test item 0,039   - 108,6 culture was not continued## 94,5 culture was not continued##
Test item 0,079   - 84,6 culture was not continued## 87,2 culture was not continued##
Test item 0,160   - 82,9 culture was not continued## 76,0 culture was not continued##
Test item 0,32   - 88,8 84 .7 99,6 11,0 0,5 79,5 77,8 96,3 16,4 4,5
Test item 0,63   - 83,0 93 .5 104,4 15,9 0,7 73,4 89,4 111,7 10,5 2,9
Test item 1,3   - 73,3 77 .8 109,3 11,3 0,5 76,2 90,7 99,1 7,4 2,1
Test item 1,9   - 57,9 69 .7 112,8 12,8 0,6 46,3 95,1 137,5 4,9 1,3
Test item 2,5   - 20,0 43 .2 112,8 27,1 1,3 18,3 73,5 155,1 8,2 2,3
Experiment II / 4 h treatment          
Solvent control with DMSO     + 100,0 100,0 100,0 15,9 1,0 100,0 100,0 100,0 3,9 1,0
Positive control (DMBA) 1,1   + 61,7 70 .9 66,5 485,0 30,6 73,8 69,5 85,0 479,5 122,2
Test item 20,0   + 95,2 culture was not continued## 102,7 culture was not continued##
Test item 40,0   + 87,8 81 .3 120,0 9,3 0,6 107,1 85,4 99,5 7,3 1,9
Test item 80,0   + 89,4 95 .5 93,7 19,5 1,2 90,1 86,3 94,3 11,0 2,8
Test item 160,0   + 82,8 86 .8 98,7 10,4 0,7 95,0 80,8 101,2 20,1 5,1
Test item 240,0   + 59,6 76 .5 107,9 8,2 0,5 100,1 84,4 96,0 21,7 5,5
Test item 300,0 PS + 0,0 12 .7 81,1 30,5 1,9 99,9 80,0 90,4 13,7 3,5

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest applied concentration in the pre-tests was 5000 µg/mL. Exceedingly strong toxic effects down to the lowest concentration occurred in the absence of metabolic activation following 4 and 24 hours treatment. Therefore, this part was repeated in a second pre-experiment using lower concentrations (0.31 - 40.0 µg/mL). The dose range of the main experiments was limited by cytotoxic effects and phase separation. The test item was dissolved in DMSO. The highest tested concentrations of the main experiments were 5µg/mL withoug S9 and 300µg/mL with S9 mix for the 4h treatment and 2.5µg/ml without S9 mix for the 24h treatment.

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. An increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the second culture of the first experiment without metabolic activation at 1.3 µg/mL. In the second experiment the induction factor exceeded the threshold at 0.32 µg/mL in culture II of the second experiment without metabolic activation and at 160, 240, and 300 µg/mL in the second culture of experiment II with metabolic activation. However, these increases were based on rather low mutation frequencies of the corresponding solvent controls (5.8, 3.6, and 3.9 mutant colonies/106 cells). Furthermore, the effects were neither reproduced in the parallel cultures under identical experimental conditions nor dose dependent as indicated by the lacking statistical significance. Therefore, these increases of the induction factor were judged as biologically irrelevant fluctuation.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 3.6 up to 21.3 mutants per 106 cells; the range of the groups treated with the test item was from 4.0 up to 30.5 mutants per 106 cells.

EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.