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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
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). The study, if used in support of isophthalic acid, has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect dated 1997.
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity: Not reported

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: agar culture
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: no reported
Additional strain / cell type characteristics:
other: hypoxanthine-guanine phospho ribosyl transferase (HGPRT)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9
Test concentrations with justification for top dose:
initial assay - non-activated: 3000, 2000, 1500, 500, 250 and 125 μg/mL
initial assay - presence of S-9: 3000, 2000, 1500, 1000, 500 and 250 μg/mL
confirmatory assay - in the absence and presence of S-9: 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate (EMS) was used as the positive control in the non-activated study at a final concentration of 0.2 μg/mL. Benzo(a)pyrene (B(a)P) was used as the positive control in the S-9 activated study at a final concentration of 4 μg/mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

The testing procedure is summarized as follows.
Cultures of CHO cells were grown in the absence of antibiotics for several passages. Mycoplasma agar plates and broth tubes were inoculated directly with the culture material and separate sets of cultures were inoculated aerobically and anaerobically. The broth tubes were subcultured three times
onto agar plates. The agar plates were examined no sooner than 14 days postinoculation for the presence of mycoplasma colonies. For the Hoechst staining procedure, the culture material was directly inoculated onto Vero cell cultures. After incubation for 3-5 days, the cell cultures were stained with the DNA--=binding fluorochrome and were evaluated microscopically by epifluorescence for the presence of mycoplasma.
SELECTION AGENT (mutation assays):
Biological Reagents:
Ham's F-12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 1% penicillin-streptomycin and 1% L-glutamine (F12FBS5-Hx)
Hank's Balanced Salt Solution (HBSS), Ca + + and Mg+ + -free (CMF-HBSS)
Trypsin, 0.05%
6-Thioguanine (TG, 2-amino-6-mercaptopurine), 20 mM in 0.1 N NaOH
Cofactor pool (final concentration in S-9 mix): 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KCl), 10 mM calcium chloride (CaCl2), 10 mM magnesium chloride (MgCl2) and 50 mM sodium phosphate buffer, pH 8.0
S-9, 9000 x g supernatant of an Aroclor-1254 induced Sprague-Dawley rat liver
homogenate, lot R-413
Giemsa stain, 10% aqueous
Methanol, 95%

NUMBER OF REPLICATIONS: initial treatment - duplicates; for evaluation of cytotoxicity, the replicates from each treatment condition were subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish; For expression of the mutant phenotype, the replicates from each treatment condition were subcultured independently in F12FBS5-Hx, in duplicate, at a density of approximately 1,000,000 cells/100 mm dish; For selection of the TG-resistant phenotype, the replicates from each treatment condition were pooled and replated, in quintuplicate, at a density of 2 x 10e5 cells/100 mm dish in F12FBS5-Hx containing 10 μM TG.

NUMBER OF CELLS EVALUATED: The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (10e6 cells: 10 plates at 2 x 10e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection. and is expressed as TG-resistant mutants per 10e6 clonable cells.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The cloning efficiency of the solvent and untreated controls must be greater than 50%. The spontaneous mutant frequency in the solvent and untreated controls must fall within the range of 0-25 mutants per 10e6 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control.
Statistics:
not reported

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
An initial and confirmatory mutagenicity assay were performed. Dose levels for the CHO/HGPRT mutation assay were selected following a preliminary toxicity test. Toxicity was based upon cloning efficiency after treatment relative to the solvent control. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 5000 to 0.5 μg/mL in the toxicity test in the absence and presence of an S-9 reaction mixture. The osmolality of the highest concentration in treatment medium tested, 5000 μg/mL, was 417 mOsm/kg with a measured pH of 4.4. The pH of each treatment condition was measured and adjusted, as needed, to approximately 6.8-7.0 using 1 N NaOH. The highest soluble concentration in culture medium was approximately 1250 μg/mL, therefore, one-half of the doses selected for each assay were usually partially to completely insoluble in culture medium. The initial cytotoxicity study demonstrated large cytotoxic effects at 5000 μg/mL. This cytotoxic effect most likely resulted from the large amount of precipitate in the dosing flask since the limit of solubility in culture medium was achieved at 1250 μg/mL. All doses were prepared by combining sufficient treatment medium for the replicate flasks at each treatment condition in an appropriately sized container with the pH adjusted as necessary. The highest concentrations tested in the study required several pH adjustments at intervals during dose preparation. Multiple pH adjustments were necessary because precipitation in the treatment media caused the pH to again decline. The pH of the dosing solutions were adjusted prior to the addition of the dosing solutions to flasks containing the target cells. Dose levels of 4500, 3500, 2000, 1500, 1000 and 500 μg/mL for the non-activated study and 5000,4000,3000, 2000 and 1000 μg/mL for the activated study were selected. This study was terminated prior to selection due to excessive toxicity exhibited at most of the treatment levels tested. New doses were selected for the initial mutagenesis assay. These dose levels were 3000, 2000, 1500, 500, 250 and 125 μg/mL in the absence of S-9 and 3000, 2000, 1500, 1000, 500 and 250 μg/mL in the presence of S-9. The doses selected for the confirmatory mutagenesis assay were 4000, 3500, 3000, 2000, 1500 and 1000 μg/mL in the absence and presence of S-9. Inconsistencies in the cytotoxicity results were noted between replicate treatments. The results from this study were not reported and an additional study was performed using the dose levels of 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL in the absence and presence of S-9. Due to inconsistent results between the replicate flasks of the 4000 μg/mL treatment in the non-activated study, the plate counts for the "B" treatment were not included in any of the calculations performed for concurrent cytotoxicity or mutation frequency for this treatment condition.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Additional tables containing cytotoxicity and mutagenicity data are in the attached pdf document.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

This study and the conclusions which are drawn from it fulfill the quality criteria (validity, reliability, repeatability).

Under the conditions of these mutagenicity tests, test substance was negative in both the absence and presence of exogenous metabolic activation.
Executive summary:

The test substance was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The initial assay was conducted at dose levels of 3000, 2000, 1500, 500, 250 and 125 μg/mL in the non-activated study and at 3000, 2000, 1500, 1000, 500 and 250 μg/mL, in the presence of S-9. The confirmatory assay was conducted at dose levels of 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL in both the absence and presence of S-9. Under the conditions of these mutagenicity tests, test substance was negative in both the absence and presence of exogenous metabolic activation.