Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome breakage (structural chromosomal aberrations) and misdistribution of chromosomes
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:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
(2014)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

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

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: Dulbecco's Modified Eagle Medium/Ham's F12 supplemented with 200 mM GlutaMAX TM, penicillin/streptomycin 100 U/mL/100 µg/mL, PHA 3 µg/mL, 10 % fetal bovine serum, 10 mM HEPES, and heparin 125 U.S.P.-U/mL.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the liver of Phenobarbital/beta-naphthoflavone induced rats.
Test concentrations with justification for top dose:
Exp. I, pulse exposure: 0.8, 1.5, 3.0, 6.0, 12.0, 24.1, 48.2, 96.4, 192.8, 385.5, 771, 1542 µg/mL (without and with S9 mix), repeat without S9 mix
Exp. II, continuous exposure: 3.0, 6.0, 12.0, 24.1, 48.2, 96.4, 192.8, 385.5, 771, 1542 µg/mL (solely without S9 mix)

The following concentrations were selected for reading: pulse exposure, without S9 mix: 96.4, 192.8, and 385.5 µg/mL; pulse exposure, with S9 mix: 48.2, 96.4, and 192.8 µg/mL; continuous exposure, without S9 mix: 48.2, 96.4, and 192.8 µg/mL.
Vehicle / solvent:
EGDE (Ethylene glycol dimethylether)
Controls
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin
Remarks:
without metabolic activation: Mitomycin C (1 µg/mL) and Demecolcin (150 ng/mL, for continuous exposure); with metabolic activation: cyclophosphamide (17.5 µg/mL)
Details on test system and experimental conditions:
Blood samples were drawn from healthy non-smoking donors not receiving medication. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with phytohemeagglutinine (PHA) and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. The lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.

Pulse exposure
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added. After 4 hrs the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium for a 16-hour recovery period. After this period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

Continuous exposure (without S9 mix)
About 48 hrs after seeding 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration. The culture medium was replaced with complete medium (with 10 % FBS) containing the test item. After 20 hours the cells were gently centrifugated, separated from the supernatant, washed and resuspended in complete culture medium. Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate.

DOSE SELECTION
With regard to the molecular weight of the test item, 1542.0 µg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 0.8 to 1542.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, phase separation of the test item was observed at the end of treatment at 771.0 µg/mL and above in the absence and at 1542.0 µg/mL in the presence of S9 mix.
Clear toxic effects were observed after 4 hours treatment with 771.0 µg/mL and above in the absence of S9 mix and with 385.5 µg/mL and above in the presence of S9 mix.
Dose selection of Experiment II was based on test item toxicity and the occurrence of phase separation. There 1542.0 µg/mL were chosen as top treatment concentration for continuous exposure in the absence of S9 mix.
Evaluation criteria:
A test item is considered to be negative if:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

A test item is considered to be positive if:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data
Statistics:
Statistical significance will be confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results
Species / strain:
lymphocytes: human
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:
No biologically relevant increase in the number of cells carrying micronuclei was observed, neither in the absence nor in the presence of S9 mix. The micronucleus rates of the cells after treatment with the test item (0.35 – 1.05 %) surpassed the range of the solvent controls (0.30-0.45 %) as well as the negative controls (0.40- 0.55 %), but were within the range of the laboratory historical control values for organic solvents other than EGDE. The positive controls used (Demecolcin, MMC or CPA) showed distinct increases in cells with micronuclei.
Thus, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH or osmolality: No relevant influence on osmolarity or pH was observed.
- Other confounding effects: Phase separation of the test item was observed at the end of treatment at 771.0 µg/mL and above in the absence and at 1542.0 µg/mL in the presence of S9 mix.

ADDITIONAL INFORMATION ON CYTOTOXICITY
In this study, at both treatment intervals, in the absence at a concentration at 771.0 µg/mL and above as well as in the presence of S9 mix at a concentration of 385.5 µg/mL, a biologically relevant cytotoxicity indicated by not evaluable concentrations could be observed.
Remarks on result:
other: strain/cell type: human lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

The substance was assessed for its potential to induce micronuclei in human lymphocytes in vitro in accordance to OECD Guideline 487. In two independent experiments the cells were exposed either for 4 hours (pulse exposure) with or without S9 mix, or for 20 hours (continuous exposure) without S9 mix. The highest concentration applied (1542.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

In this study, at both treatment intervals, a biologically relevant cytotoxicity indicated by not evaluable concentrations could be observed (in the absence of S9 mix at a concentration of 771.0 μg/mL and above, in the presence of S9 mix at a concentration of 385.5 μg/mL). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of cells after treatment with the test item (0.35-1.05 %) surpassed the range of the solvent controls (0.30-0.45 %) as well as the negative controls (0.40- 0.55 %), but were within the range of the laboratory historical control values for organic solvents other than EGDE.

The positive controls used (Demecolcin, MMC or CPA) showed distinct increases in cells with micronuclei.

Thus, the test item was considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest evaluable concentration.