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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 Feb 2010 - 30 Jun 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid/colorless, clear
- Storage condition of test material: ambient (room temperature)/ under light exclusion

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL) and 1% (v/v) amphotericin B (250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
1st Experiment (without light protection)
(without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL)
(with S9 mix: 0; 312.5; 625; 1 250; 2 500; 3750; 5000 μg/mL)
2nd Experiment (without light protection)
without S9 mix: 0; (5); (10); (20); 40; 60; 80 μg/mL
with S9 mix: 0; (78.1); (156.3); 312.5; 625; 1250; (2500); (3750) μg/mL
3rd Experiment (under light protection)
without S9 mix: 0; 6.3; 12.5; 25; (50); (100), (200) μg/mL
with S9 mix: 0; 156.3; 312.5; 625; (1250); (2500); (5000) μg/mL
(test groups in brackets were not evaluated)
Vehicle / solvent:
acetone 1% (v/v)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS: 500 μg/mL; CPP: 0.5 μg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4h (both with and without S9)
- Expression time (cells in growth medium): 14 h (both with and without S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 18 h (both with and without S9)

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): 7.5% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS: All cultures were prepared in duplicate.

NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations. Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; a mitotic index based on 1 000 cells/culture was determined for all evaluated test groups in all cytogenetic experiments.

OTHER EXAMINATIONS:
Aneuploid and polyploid cells were recorded separately.
Evaluation criteria:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups, and if cells had 20 – 22 chromosomes, they were analyzed for structural chromosome aberrations.
The test substance is considered as “positive” if the following criteria are met:
• A statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps).
• The number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range.
A test substance generally is considered as “negative” if the following criteria are met:
• The number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program system (BASF SE). The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤ 0.05, ** p ≤ 0.01) are printed in the tables.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not influenced by test substance treatment
- Effects of osmolality: not influenced by test substance treatment
- Precipitation: In the main experiments, test substance precipitation in culture medium at the end of exposure period was observed from 625 μg/mL onward only in the presence of S9 mix in all experimental parts.

RANGE-FINDING/SCREENING STUDIES:
In the pretest performed without light protection the pH was not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. In addition, no test substance precipitation in the vehicle acetone was observed up to the highest required concentration of 5000 μg/mL (stock solution). In culture medium test substance precipitation occurred at 625 μg/mL and above 4 hours after start of treatment in the absence and the presence of S9 mix as well as at 18 hours continuous treatment in the absence of S9 mix. At these concentrations an oily film of the test substance was observed on surface of the culture medium.
After 4 hours treatment in the absence of S9 mix cytotoxicity indicated by reduced cell numbers of below 50% of control was observed at 78.1 μg/mL and above. In addition, in the presence of S9 mix, clearly reduced cell numbers were observed after treatment with 5000 μg/mL. Besides, in the pretest with 18 hours continuous treatment in the absence of S9 mix, the cell numbers were clearly reduced after treatment with 156.3 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
Second experiment: In the presence of metabolic activation after 4 hours treatment a statistically significant increase in the number of chromosomally damaged cells was observed at the highest scorable concentration of 1250 μg/mL (18% aberrant metaphases excl. gaps). This value clearly exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).
Third experiment: In the absence and presence of S9 mix a statistically significant increase in the number of aberrant metaphases was observed at all concentrations scored for cytogenetic damage performed under light protection conditions. In both experimental parts most values exceeded our historical negative control data range (0.0% – 5.5% aberrant metaphases excl. gaps).

CELL MORPHOLOGY
Cell attachment was influenced from 40.0 μg/mL onward in the 1st or 2nd Experiment without S9 mix. When applying light protection in the 3rd Experiment the cell attachment was slightly reduced from 12.5 μg/mL onward in the absence of S9 mix. After the addition of S9 mix cell morphology was influenced from about 625 μg/mL onward in all experiments performed with or without light exclusion.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix 4 hours after treatment in the 2nd and 3rd Experiment, no suppression of the mitotic activity was observed at any dose analyzed for chromosomal aberrations. In the 2nd Experiment in the absence of metabolic activation after 4 hours treatment up to 80 μg/mL no reduction of mitotic activity was obtained. In the 3rd Experiment in the absence of metabolic activation the slides from 50 μg/mL onward were not scorable due to low metaphase numbers. Besides, in the presence of S9 mix in the 2nd and 3rd Experiment after 4 hours treatment the concentrations showing clearly reduced mitotic indices were not scorable for cytogenetic damage due to low metaphase numbers and/or poor metaphase quality (from 2 500 μg/mL or 1 250 μg/mL onward, respectively).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system. The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either.

Summary table – experimental parts without S9 mix

Exp. Schedule Test groups S9 mix P Genotoxicity Cytotoxicity*
Exposure/ preparation period Aberrant cells [%] Polyploidy cells [%]
Incl. gaps# Excl. gaps# With exchanges Cell no. [%] Mitotic index [%]
2 4/18 h Vehicle control1 - - 7 3 0.5 0 100 100
5.0 mg/mL - - n.d. n.d. n.d. n.d. 98 n.d.
10.0 mg/mL - - n.d. n.d. n.d. n.d. 85 n.d.
20.0 mg/mL - - n.d. n.d. n.d. n.d. 81.8 n.d.
40.0 mg/mL - - 5 3.5 1 0 82.9 111.6
60.0 mg/mL - - 4.5 3.5 1 0 81.8 105.5
80.0 mg/mL - - 7 5.5 3.5 0 38.7 103.4
Positive control2x - - 21.0s 18.0s 10.0s 0 n.t. 103.4
     Performance under light protection
2 4/18 h Vehicle control1 - - 4 1.5 0.5 0 100 100
6.3 mg/mL - - 8.5s 5.5s 3 1 74.8 89.4
12.5 mg/mL - - 13.5s 9.5s 7.5s 0.5 47.6 105.3
25.0 mg/mL - - 14.5s 10.0s 6.5s 0 46.5 104.6
50.0 mg/mL - - n.s. n.s. n.s. n.s. 4.1 n.s.
100.0 mg/mL - - n.s. n.s. n.s. n.s. 3.2 n.s.
200.0 mg/mL - - n.s. n.s. n.s. n.s. 4.8 n.s.
Positive control2x - - 20.0s 17.0s 10.0s 0 n.t. 82.8

Summary table – experimental parts with S9 mix

Exp. Schedule Test groups S9 mix P Genotoxicity Cytotoxicity*
Exposure/ preparation period Aberrant cells [%] Polyploidy cells [%]
Incl. gaps# Excl. gaps# With exchanges Cell no. [%] Mitotic index [%]
2 4/18 h Vehicle control1 + - 5 3.5 1.5 0 100 100
78.1 mg/mL + - n.d. n.d. n.d. n.d. 88.9 n.d.
156.3 mg/mL + - n.d. n.d. n.d. n.d. 89.8 n.d.
312.5 mg/mL + - 7.5 5 3.5 0 78.5 103
625.0 mg/mL + - 6 4.5 3 0 78.5 79.2
1250.0 mg/mLx + + 18.0s 18.0s 16.0s 0 55.2 81.7
2500.0 mg/mL + + n.s. n.s. n.s. n.s. 56.7 29.2
3750.0 mg/mL + + n.s. n.s. n.s. n.s. 27.4 n.s.
Positive control3x + - 27.0s 25.0s 10.0s 0 n.t. 60.4
     Performance under light protection
2 4/18 h Vehicle control1 + - 4.5 2 0.5 0 100 100
156.3 mg/mL + - 11.5s 9.0s 4.0s 0.5 87.1 84.8
312.5 mg/mL + - 13.0s 9.0s 4.0s 0.5 83 99
625.0 mg/mLx + + 31.0s 30.0s 23.0s 0 73 85.3
1250.0 mg/mL + + n.s. n.s. n.s. n.s. 48.8 40.6
2500.0 mg/mL + + n.s. n.s. n.s. n.s. 51.2 n.s.
500.0 mg/mL + + n.s. n.s. n.s. n.s. 41.4 n.s.
Positive control3x + - 26.0s 22.0s 12.0s 0 n.t. 77.7

P Precipitation occured at the end of exposure period

* Relative values compared with the respective vehicle control

# Inclusive cells carrying exchanges

s Aberration frequency statistically significant higher than corresponding control values

x Evaluation of a sample of 100 metaphase only due to strong clastogenicity

1 Acetone 1% (v/v)

2 EMS500 μg/mL

3 CPP 0.5mg/mL

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity and/or poor metaphase quality

n.t. Not tested

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that the test article is a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.
Executive summary:

The test substance was assessed for its potential to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) in V79 cells in vitro both in the absence and the presence of a metabolizing system. During the experimental phase of this study the lab was informed by the sponsor that the test substance is an UV-curing agent. Therefore, it is light sensitive and has to be stored and handled protected from light. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, and taking into account the cytotoxicity actually found in the main experiments, the following concentrations were tested and the test groups in bold type were evaluated:

1st Experiment (without light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 312.5; 625; 1250; 2500; 3750; 5000 μg/mL

The 1st Experiment with and without S9 mix had to be repeated due to severe cytotoxicity (strongly reduced mitotic rates) and partly poor metaphase quality.

2nd Experiment (without light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 5; 10, 20; 40; 60; 80 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 78.1; 156.3; 312.5; 625; 1250; 2500; 3750 μg/mL

The 2nd Experiment with and without S9 mix had to be repeated due to an update on storage conditions and handling instructions indicating protection from light.

3rd Experiment (under light protection)

4-hour exposure, 18-hour sampling time, without S9 mix: 0; 6.3; 12.5; 25; 50; 100, 200 μg/mL

4-hour exposure, 18-hour sampling time, with S9 mix: 0; 156.3; 312.5; 625; 1250; 2500; 5000 μg/mL

A sample of 100 metaphases for each culture was analyzed for chromosomal aberrations, except for the positive control cultures and the concentrations 1250 μg/mL and 625 μg/mL in the 2nd and 3rd Experiment, respectively, where only 50 metaphases were scored due to clearly increased aberration rates. The vehicle controls gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive control substances, EMS and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. On the basis of the results of the present study, the test substance caused a statistically significant and dose-dependent increase in the number of structurally aberrant metaphases incl. and excl. gaps after 4 hours exposure without light protection in the presence of metabolic activation. in a single experiment (2nd Experiment). In the 3rd Experiment under light protection a statistically significant and biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps was obtained after 4 hours exposure either without S9 mix or after adding a metabolizing system (see Table 1, page 14 – 15). The observation of clastogenicity in the 2nd Experiment was confirmed in the 3rd Experiment (performed under light protection conditions). Therefore, it can be assumed that the toxic effect obtained in the 2nd Experiment was not induced by light absorption. No relevant increase in the frequency of cells containing numerical chromosome aberrations was demonstrated either. Thus, under the experimental conditions described, the test article is considered to have a chromosome-damaging (clastogenic) effect under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.