[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Federal Register 61: 18198-18202, April 24, 1996

GLP compliance:

yes

Remarks:

US FDA 21 CFR 58, US EPA 40 CFR 792, Japanese GLP Standards, and OECD

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): C-4000
- Physical state: liquid
- Analytical purity: 86.3 +/- 0.7%
- Purity test date: 2008-01-01 through 2008-01-25
- Lot/batch No.: 51V034K7
- Expiration date of the lot/batch: 2008-02-01
- Storage condition of test material: 2-8 degrees; protected from light

Method

Target gene:

HGPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: F12 medium without hypoxanthine, supplemented with 5% dialyzed fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes, for each frozen batch
- Periodically checked for karyotype stability: yes, for each frozen batch
- Periodically "cleansed" against high spontaneous background: yes, with HAT (hypoxanthine, aminopterin and thymidine). Cells used in the mutation assay will not exceed four subpassages from time of cleansing.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 ug/mL
Cytotoxicity testing: 5, 10, 15, 25, 50 ug/mL
Mutation assay: 5, 10, 15, 25, 50 ug/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility of the test article and compatibility with the target cells.

Controls

Untreated negative controls:

yes

Remarks:

Vehicle used as negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulfonate was used as the positive control for the non-activates test system and benzo(a)pyrene was used as the positive control for the S-9 activatetd system.

Migrated to IUCLID6: and benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Cells seeded 18-24 hours before treatment
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 7-9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 to 18 days from treatment to staining of mutant colonies.


SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2 at each test article concentration.


NUMBER OF CELLS EVALUATED: 106 cells treated at each concentration, 2 x 106 cells seeded for mutant selection.


DETERMINATION OF CYTOTOXICITY
- relative cloning efficiency


OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Other: mutant colonies (6-TG-resistant)

Evaluation criteria:

The cloning efficiency of the solvent control must be greater than 50%. The spontaneous mutant frequency in the solvent control must fall within the range of 0 25 mutants per 106 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control and must exceed 40 mutants per 106 clonable cells. There must be at least four analyzable test article concentrations with mutant frequency data.

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None noted.
- Effects of osmolality: No effect, the solvent and treat cultures had virtually the same osmolality.
- Evaporation from medium: None noted.
- Water solubility: DMSO was the solvent for the test article
- Precipitation: There was visible precipitate in the treatment medium at concentrations ≥ 50 µg/mL.
- Other confounding effects: N/A


RANGE-FINDING/SCREENING STUDIES: Range-finding assay done to determine concentrations for the mutation assays.


COMPARISON WITH HISTORICAL CONTROL DATA: Yes, controls were within range.


ADDITIONAL INFORMATION ON CYTOTOXICITY: N/A

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

All criteria for a valid study as described in the protocol were met. C-4000 was negative in the CHO/HGPRT Mutation Assay under the conditions of this study.

Executive summary:

The test article, C-4000, was tested in the CHO/HGPRT Mutation Assay in the absence and presence of metabolic activation using Aroclor-induced rat liver S9. The preliminary toxicity assay was used to establish the concentration range for the initial mutagenesis assay.  The initial and independent repeat mutagenesis assays were used to evaluate the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was chosen as the solvent for the test article based on information from the Sponsor and compatibility with the target cells. The test article was soluble in DMSO at a concentration of 500 mg/mL, the maximum concentration prepared for the preliminary toxicity assay.

In the preliminary toxicity assay, the maximum concentration of C-4000 (Lot51V034K7)tested was 5000 µg/mL. There was visible precipitate in the treatment medium at concentrations ≥ 50 µg/mL. Selection of concentrations for the mutagenesis assay was based on the cloning efficiency relative to the solvent control and precipitation profile. No substantial toxicity, i.e., cloning efficiency ≤ 50% of the solvent control, was observed with or without S9 activation. Based on these findings, the concentrations chosen for the mutagenesis assay ranged from 2.5 to 50 µg/mL for both the non-activated and S9-activated cultures.

 

In the initial mutagenesis assay, no positive responses, i.e., treated cultures with mutant frequencies >40 mutants per 10⁶ clonable cells, were observed. Visible precipitate was observed in treatment medium at 50 µg/mL. No toxicity, i.e., cloning efficiency ≤50% of the solvent control, was observed.

In the independent repeat assay, no positive responses were observed. Visible precipitate was observed in treatment medium at 50 µg/mL. No toxicity was observed.

C-4000 was negative in the CHO/HGPRT Mutation Assay under the conditions of this study.