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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - 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 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-(Dimethylamino)-2-methyl-1-propanol
- Analytical purity: 79.0 ± 0.1%
- Impurities (identity and concentrations): 19.5 ± 0.12% water
- Lot No.: WI024801S1

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: cultured rat (Crl:CD(SD)) lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium with 25 mM HEPES (GIBCO, Grand Island, New York) supplemented with:
- 10% heat-inactivated foetal bovine serum (GIBCO),
- antibiotics and antimycotics (Fungizone 0.25 µg/mL; penicillin G, 100 u/mL; and streptomycin sulphate, 0.1 mg/mL; GIBCO)
- 30 µg/mL PHA-P (HA16, Murex Diagnostics Ltd., Dartford, England)
- 2 mM L-glutamine (GIBCO).
Cultures were initiated by inoculating approximately 0.5 mL of whole blood/5 mL of culture medium.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Crl:CD(SD) rats treated with 500 mg/kg bw Aroclor 1254
Test concentrations with justification for top dose:
Experiment I - range-finder experiment:
- Short treatment (4 h): 18.3, 36.6, 73.3, 146.5, 293, 586, and 1172 µg/mL with and without metabolic activation
- Continuous treatment (24 h): 9.2, 18.3, 36.6, 73.3, 146.5, 293, 586, and 1172 µg/mL without metabolic activation

Experiment II - cytotoxicity test:
- Short treatment (4 h): 73.3, 146.5, 293, #586, #879, and #1172 µg/mL with and without metabolic activation
- Continuous treatment (24 h): 73.3, 146.5, 293, #586, #879, and #1172 µg/mL without metabolic activation
# concentrations selected for determination of chromosomal aberrations and the incidence of polyploidy

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
mitomycin C (MMC), 0.5 µg/mL (4 h treatment) or 0.05 and 0.075 µg/mL (24 h treatment), -S9; cyclophosphamide (CP), 2 and 4 µg/mL (4 h treatment), +S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: experiment I and II: 4 h (±S9), 24 h (-S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 ± 1 h

SPINDLE INHIBITOR: colcemid, 1 µg/culture
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicate in two independent experiments

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: visual assessment and evaluation (experiment I); mitotic index of 1000 cells per culture (experiment II)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, in 100 metaphases per culture
Evaluation criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the solvent controls. The aberration frequency in the solvent control should be within reasonable limits of the laboratory historical values. A test chemical was considered positive in this assay if it induced a significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells and cultures was outside the recent historical solvent control range.
Statistics:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyse the frequencies of aberrant cells. An overall Chi-square statistic was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e. control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971). Polyploid cells were analysed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analysed separately based on the presence or absence of S9 and based on the exposure time.

Results and discussion

Test results
Species / strain:
lymphocytes: cultured rat (Crl:CD(SD)) lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h: minimal cytotoxicity ( no clear dose-response); 24 h: ≥ 586 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: since an appreciable change in the pH of the treatment medium was observed after the addition of the test material, 1 N HCl was used to adjust the pH of the 4 highest test concentrations (i.e., 293, 586, 879, and 1172 µg/ml) to the acceptable range of pH 6.8 to 7.6.
- Other confounding effects: in experiment I, colcemid was added 2 h after the time point specified in the study protocol and the OECD test guideline. Thus, evaluation of cytotoxicity after treatment with the test substance was only performed by visual assessment and based on this assessment the experiment was repeated.

RANGE-FINDING/SCREENING STUDIES: due to the delayed treatment with colcemid in the first experiment, only visual assessment of cytotoxicity was performed after treatment of lymphocytes with the test substance in the presence or absence of metabolic activation. Based on this screening procedure, concentrations of 73.3, 146.5, 293, 586, 879, and 1172 µg/mL were chosen for cytotoxicity analysis in experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA: the frequencies of aberrant cells observed in the test material treated cultures were within the laboratory historical background range of the solvent control.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the absence of S9 mix, the cell cultures displayed little to no toxicity with mitotic indices ranging from 90.2 to 100.6% compared to the solvent control after short-term treatment (4 h). In the presence of S9 mix, the mitotic indices of the treated cultures ranged from 77.3 to 95.7% as compared to the solvent control after 4 h exposure. Cultures treated continuously for 24 h in the absence of S9 activation had minimal toxicity with mitotic indices ranging from 78.4 to 109.8% relative to the solvent control.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

DETERMINATION OF OSMOLALITY

The osmolality of treatment medium containing approximately 1172 µg/mL of the test material (osmolality = 281 mOsm/kg water) and medium containing 1% distilled water as solvent (271 mOsm/kg water) were comparable. The adjustment of pH of the 4 highest concentrations (i.e., 293, 586, 879, and 1172 µg/mL) did not unduly alter the osmolality.

 

ANALYTICAL VERIFICATION OF TEST SUBSTANCE SOLUTIONS

Analytically detected concentrations of the test material in the stock solutions varied from 96.4 to 108.8% of the target concentrations, and thus verified that concentrations used for treatment were within acceptable values.

DETERMINATION OF CHROMOSOMAL ABERRATIONS AND POLYPLOIDY

Table 1. Test results of experiment II

Test item

Concentration in µg/mL

Mitotic Index in %

Total aberrant cells in %*

Incidence of polyploidy in % (based on 100 metaphases)

Exposure period 4h, fixation time 24h, without S9 mix

Distilled water

1.0% (v/v)

100

0.5

0.0

MMC

0.5

92

11

0.5

Test substance

586

100.6

0.5

1.5

879

90.2

0.0

0.5

1172

93.9

0.5

1.0

Exposure period 4h, fixation time 24h, with S9 mix

Distilled water

1.0% (v/v)

100

1.0

1.0

CPa

4

57.4

28.0

0.5

Test substance

586

87.9

1.0

0.5

879

88.7

1.0

0.5

1172

79.4

1.5

1.0

Exposure period 24h, fixation time 24h, without S9 mix

Distilled water

1.0% (v/v)

100

0.5

0.0

MMCb

0.075

78.4

13.3

0.0

Test substance

586

88.2

0.0

0.5

879

78.4

0.0

1.0

1172

78.4

0.0

0.5

MMC: mitomycin C; CP: cyclophosphamide (positive controls)
*excluding gaps
aa total of 100 cells was evaluated
ba total of 150 cells was evaluated

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative