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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
Study period:
Nov./Dec. 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
HPRT assay testing for reversion to resistance to the purine-analogue, 6-thioguanine, as result of a mutation in the X-chromosome-linked hypoxanthine-guanine phosphoribosyl transferase (HPRT).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report) = Cab-O-Sil EH-5

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham´s F-12 medium without hypoxanthine with 5 % FBS, 1 % penicilin-streptomycin and 1 % L-glutamine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
-Source: A. Hsie, Oak Ridge National Laboratories, directly received in frozen state
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 (adult male SD rats)
Test concentrations with justification for top dose:
10, 50, 100, 150, and 250 µg/mLNgative (without S9) and 100, 200, 300, 400, and 500 µg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, 1 % final concentration
- Justification for choice of solvent/vehicle: No data
Controls
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate (without S9) and benzo(a)pyrene (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 18 - 24 hours at 37 +-1 °C, 5 x10^5 cell/25 mL flask in 5 +- 1% CO2 atmosphere
- Exposure duration: 5 h (without and with S9)
- Expression time (cells in growth medium): Post exposure: 18 - 24 h, followed by 7 - 9 days including subculturing of 2 - 3 day intervals
- Selection time (if incubation with a selection agent): In the presence of 6-thioguanine, 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 - 17 days

STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2 x10^5 cells /100 mm dish (five-fold) = in total 10^6 cells per concentration

DETERMINATION OF CYTOTOXICITY
- in parallel as cloning efficiency (triplicates with each 100 cells/60 mm dish)


Evaluation criteria:
A response was not considered positive unless the mutant frequency exceeded 20 mutants per 10^6 clonable cells.
Significant if twice that of background and at least 11 mutants per 10^6 cells above background (solvent and untreated control).
Positive if dose-dependen increase in mutant frequency combined with significant increase at one or more test concentrations.
Suspect if no dose-response.
Statistics:
Frequency of spontaneous mutations (if showing wide variation): C.I. (Conf. Interval) calculated by application of the one-sided student´s test from the historical background rate (upper limit of C.I.: 11 spontaneous mutants/10^6 cells).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: Not pronounced in the mutation test, while a concurrent cytotoxicity test showed considerable inhibition of the cloning efficiency over the same dose ranges.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: 9 concentrations between 0.1 and 500 µg/mL tested with and without S9, toxicity based on colony-forming efficiency

Any other information on results incl. tables

From Report Tab. 3 and 4

With S9 (all data relating to x mutants/106 cells)

negative controls: 1 and 7.2

positive control: 351

treated cells: no dose response

1 and <1 at higher dose levels (100 to 250 µg/mL) with cytotoxicity noted at 250 µg/mL.

7.3 and 10.3 at the lower dose levels (10 and 50 µg/mL, respectively).

Without S9 (all data relating to x mutants/106 cells)

negative controls: 0.8 and 8.0

positive control: 184

treated cells: no dose response

<1 to 8.2

no pronounced cytotoxcity noted at any dose level

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative