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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:
15 May 2102 - 19 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance on Specific Aspects of Regulatory Genotoxicity Tests of Pharmaceuticals
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Name of test material (as cited in study report): Jeffcat ZR-50
- Substance type: Amber liquid
- Physical state: Liquid
- Analytical purity: 100%
- Lot/batch No.: PFW100119
- Storage condition of test material: Room temperature, stored protected from light

Method

Target gene:
HGPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
CHO cells were cleansed in medium supplemented with hypoxanthine, aminopterin and thymidine (HAT).
F12FBS5+Hx medium (seeding and treatment, cultering after treatment)
F12FBS5-Hx medium is Ham's F12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 100 units penicillin/mL, 100 µg streptomycin/mL and 2 mM L-glutamine/mL.
- Properly maintained: yes
- Cells used in each mutation assay were within four subpassages from cleansing in order to assure karyotypic stability
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity assay: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 2000 µg/mL
Initial concurrent cytotoxicity test: 250, 500, 1000, 1500, 1750 and 2250 µg/mL (non-activated cultures) and 250, 500, 1000, 1500 and 2500 µg/mL (S9-activated cultures)
Mutation assay: 250, 500, 1000, 1500 and 2500 µg/mL (activated study)
Repeat concurrent cytotoxicity test: 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL (non-activated cultures)
Mutation assay: 500, 750, 1000, 1250, 1500 and 2000 µg/mL (non-activated study)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile distilled water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
4 µg/mL in S9 activated cultures
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
0.2 µg/mL in non-activated cultures
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 2 days (7 to 9)
- Selection time (if incubation with a selection agent): 3 days incubation
- Fixation time (start of exposure up to fixation or harvest of cells): no data

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 2 x 1E06 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
- The test article was considered to induce a positive response if there was a concentration-related increase in mutant frequencies with at least two consecutive concentrations showing mutant frequencies of > 40 mutants per 1E06 clonable cells
- If a single point above 40 mutants per 1E06 clonable cells was observed at the highest concentration, the test article was considered equivocal
- If no culture exhibited a mutant frequency of > 40 mutants per 1E06 mutants per 1E06 clonable cells, the test article was considered negative.

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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: In order to reach pH 7, the pH of the five to eight highest concentrations was adjusted, using 1N HCl prior to adding S9 or target cells to the treatment medium.
- Effects of osmolality: The osmolality of the solvent control was 280 mmmol/kg and the osmolality of the top concentration, 2500 µg/mL, was 300 mmol/kg (preliminary toxicity assay).
- Precipitation: In the preliminary toxicity assay, no visible precipitate was observed in the treatment medium at the beginning or end of treatment.
- Water solubility: The test article formed a clear solution in water at approximately 25 mg/mL in the solubility test.

RANGE-FINDING/SCREENING STUDIES:
Cloning efficiency relative to the solvent controls (relative cloning efficiency) at 2500 µg/mL was 0% without activation and 100% with S9 activation. Based on the results of the toxicity test, the concentrations chosen for the initial mutagenesis assay ranged from 250 to 2250 µg/mL for the non-activated cultures and from 250 to 2500 µg/mL for the S9-activated cultures.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the solvent and positive controls are situated within the ranges of the historical control data (data from 2009-2011).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the initial mutagenesis assay, no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Relative cloning efficiency was 60% at a concentration of 1500 µg/mL and < 10% at concentrations >= 1750 µg/mL in the non-activated system and 92% at the high concentration in the S9-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells. The non-activated portion failed due to a lack of cultures with between 10 and 20% relative cloning efficiency. The mutagenesis assay was repeated in the absence of S9 activation using concentrations from 500 to 1750 µg/mL.

The second and third trials of the mutagenesis assay in the absence of S9 activation failed due to the presence of contamination in the cultures. Based on the toxicity results from the first and second trial of the mutagenesis assay, the concentrations chosen for the third and fourth trials of the mutagenesis assay in the absence of S9 activation ranged from 500 to 2500 µg/mL.

Fourth trial: no visible precipitate was observed in the treatment medium at the beginning or end of treatment. Cultures treated with concentrations of 500, 750, 1000, 1250, 1500, 2000 and 2250 µg/mL were cloned for concurrent cytotoxicity. Relative cloning efficiency was 54% at a concentration of 1500 µg/mL and < 10% at concentrations >= 2000 µg/mL in the non-activated system. None of the treated cultures exhibited mutant frequencies of greater than 40 mutants per 1E06 clonable cells.
Remarks on result:
other: all strains/cell types tested

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met. The results of the CHO/HGPRT Mutation Assay indicate that, under the conditions of this study, the test substance was concluded to be negative with or without metabolic activation.