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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: HPRT

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
MEDIA USED
- Properly maintained:yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
rat S9 mix (phenobarbital and ß-naphthoflavone induced) from male Wistar rat livers
Test concentrations with justification for top dose:
1st Exp.: 3.13; 6.25; 12.50; 25.00; 50.00; 100.00; 200.00 µg/mL (with and without S9 mix)
2nd Exp.: 4.69; 9.38; 18.75; 37.50; 75.00; 150.00 µg/mL (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 20 - 24 h
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): from day 16

SELECTION AGENT (mutation assays): 6-thioguanine (10 μg/mL)

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried
out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

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:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced relative survival of about or below 20% of the respective negative control values were observed in the 1st and 2nd Experiment in the absence of S9 mix at least at the highest applied concentrations. In the absence of S9 mix , there was a distinct decrease in the number of colonies at 50 μg/mL (RS: 38.0%) after an exposure period of 4 hours in the 1st Experiment. Higher concentrations were not seeded for cloning efficiency 1 because cell densities were severely decreased at the end of exposure period. Additionally, in the 2nd Experiment strong cytotoxicity was determined at 150 μg/mL (RS: 3.7%). The cell density was distinctly reduced at the highest applied concentration of 150 μg/mL. In contrast, in the presence of S9 mix, there was no decrease in the number of colonies up to the highest applied concentration in the 1st and 2nd Experiment (200 and 150 μg/mL, respectively). The cell densities were not effected by test substance exposure.

Applicant's summary and conclusion

Conclusions:
Thus, in the absence and the presence of metabolic activation, C9-11 Methacrylate is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.