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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-11-28 - 2012-06-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 487: In Vitro Mammalian Cell Micronucleus Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): PEROXAN BCC
- Physical state: white powder
- Analytical purity: 98.5%
- Lot/batch No.: 108104
- Expiration date of the lot/batch: 01 Feb 2012
- Stability under test conditions: unknown
- Storage condition of test material: max. 15 °C ; control temperature: + 30 °C ; Emergency temperature: +35 °C
The test item was stored in closed vessel kept at 2 to 8 °C.

Method

Species / strain
Species / strain / cell type:
lymphocytes: human periheral blood obtained by a donor
Details on mammalian cell type (if applicable):
Human peripheral blood lymphocytes were obtained from an adequate donor (healthy, non-smoking, no known recent exposures to genotoxic chemicals or radiation). Blood samples were drawn by venous puncture and collected in heparinized tubes. Blood cul-tures were set up within 24 hours after sample collection.
- Type and identity of media:







- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from the liver of male rats
Test concentrations with justification for top dose:
Exp. I: 3992.8, 1996.4, 998.2, 499.1, 249.5, 124.8, 62.4 and 31.2 μg/mL
Exp. II: 3990, 1995, 997.5, 498.8, 249.4, 124.7, 62.4 and 31.2 μg/mL
Exp. III: 39.3, 19.6, 9.8, 4.9, 2.5, 1.2, 0.6 and 0.3 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetra hydrofuran (THF) for the test material and 0.9% NaCl for the positive controls.
- Justification for choice of solvent/vehicle: based on the pre-experiment (exp. I) the test material was sufficiently soluble in THF.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
tetra hydrofurane (THF)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
With S9-Experiment I & II
-Exposure duration: 4 ± 1 h; the medium was removed, fresh medium and cytoB were added. The cells were harvested 1.5-2 cell cycles later.

Without S9-Experiment I
-Exposure duration: 4 ± 1 h; the medium was removed, fresh medium and cytoB were added. The cells were harvested 1.5-2 cell cycles later.

Without S9, extended exposure-Experiment II & III
-Exposure duration: 20 ± 2 h (1.5 - 2 cell cycles) in the presence of cytoB. The cells were harvested at the end of the exposure period.

SPINDLE INHIBITOR (cytogenetic assays): cytoB
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: at least 500 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (cytokinesis-block proliferation index)


Evaluation criteria:
The test item is considered to have no genotoxic effects if:
-the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data of the solvent control
-no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

The test item is considered to have genotoxic effects if:
-the number of micronucleated cells in all evaluated dose groups is above the range of the historical laboratory control data
-either a concentration-related increase of micronucleated cells or a statistically significant increase in the number of cells containing micronuclei is observed.
Statistics:
Statistical significance when p<0.01

Results and discussion

Test results
Species / strain:
lymphocytes:
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:
Solubility of the test substance was determined in a non-GLP pre-test. The test item was sufficiently soluble in tetrahydrofurane (THF).
The study was considered acceptable: Micronucleus induction of the negative controls was within the range of the literature data (0.1–0.8%). The positive controls showed distinct increases in the number of binucleated cells with micronuclei.
Exp. I: No cytotoxicity or precipitation in any of the concentrations tested. No micronuclei were detected, with the exception of two concentrations; however, the response was not dose-related and hence, it was considered irrelevant.

Exp. II: With S9 - no cytotoxicity or precipitation in a ny of the concentrations tested. No increase in the number of binucleated cells with miconuclei.
Without S9 (prolonged exposure) - strong cytotoxicity did not allow for further evaluation.

Exp. III: Without S9 - weak cytotoxicity seen in all concentrations. No significant increase in micronuclei.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

On the basis of this result, PEROXAN BCC does not induce genotoxicity in human lymphocytes.
Executive summary:

In a mammalian cell cytogenicity test (micronucleus test) human peripheral blood lymphocytes (obtained by a donor) were exposed to PEROXAN BCC (analytical purity: 98.5%) in tetradyrofuran (THF) in the presence and absence of mammalian metabolic activation (liver S9 mix, male rats). Prior to exposure the cells were properly cultured in the presence of phytohaemagglutinin.

Three experiments were run as follows:

Experiment I

With and without S9 mix / 4 hrs exposure: 3992.8, 1996.4, 998.2, 499.1, 249.5, 124.8, 62.4 and 31.2 μg/mL

Concentrations for micronuclei scoring: 3992.8, 1996.4 and 998.2 μg/mL

Experiment II

Without S9 mix / 20 ± 2 hrs exposure: 3990, 1995, 997.5, 498.8, 249.4, 124.7, 62.4 and 31.2 μg/mL

With S9 mix / 4 hrs exposure: 3990, 1995, 997.5, 498.8, 249.4, 124.7, 62.4 and 31.2 μg/mL

Concentrations for miconuclei scoring: 3992.8, 1996.4 and 998.2 μg/mL

Experiment III

Without S9 mix / 20 ± 2 hrs exposure: 39.3, 19.6, 9.8, 4.9, 2.5, 1.2, 0.6 and 0.3 μg/mL

Concentrations for miconuclei scoring: 19.6, 9.8 and 4.9 μg/mL

There was no evidence or a concentration related positive response of induced micronuclei in the human lymphocytes.The positive controls did induce the appropriate response.

This study is classified as acceptable.This study satisfies the requirements of test guideline OECD 487 for in vitro cytogenicity (mammalian cell micronucleus test) data.