Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Study period:
1990
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethoxymethane
EC Number:
203-714-2
EC Name:
Dimethoxymethane
Cas Number:
109-87-5
Molecular formula:
C3H8O2
IUPAC Name:
dimethoxymethane
Test material form:
other: liquid
Details on test material:
Name of test material (as cited in study report): C-01361 (=test ID of dimethoxymethane)
Methylal min 97.5% wt
Water: max 1.5% wt
Acidity, as acetic: max 0.01% wt
Aldehydes, as formaldehyde: max 0.10% wt
Methanol: max 1.0% wt

Method

Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's Nutrient Mixture F12 supplemented with L-glutamine, antibiotics and fetal bovine serum (8% by volume) was used as culture medium during experimental studies. .
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction and reaction mixture as described in "Any information on materials and methods incl. tables"
Test concentrations with justification for top dose:
RANGE FINDING TEST
Ten concentrations were used (with and without metabolic activation) for preliminary range-finding cytotoxicity test: 0.0098, 0.0195, 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0 mg/ml.

MUTATION TEST
Six concentrations were tested (with and without metabolic activation) for mutation assay: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mg/ml.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Sterile deionised water
- Justification for choice of solvent/vehicle: The test material was soluble in sterile deionised water
Controls
Untreated negative controls:
yes
Remarks:
only used in (range-finding) cytotoxicity assay
Negative solvent / vehicle controls:
yes
Remarks:
used in both cytotoxicity (range-finding) and mutation assays
True negative controls:
no
Positive controls:
yes
Remarks:
only used in mutation assay
Positive control substance:
3-methylcholanthrene
Remarks:
Migrated to IUCLID6: other: 5-bromo-2-deoxyuridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION for preliminary range-finding cytotoxicity assay
- Preincubation period: 16-18h
- Exposure duration: 4 hours at 35.5-38.5°C
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): no
- Fixation time (start of exposure up to fixation or harvest of cells): About 6 days and 4hours

DURATION for mutation assay
- Preincubation period: about 18 h
- Exposure duration: 4 hours at 35.5-38.5°C
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7-10 days
- Fixation time (start of exposure up to fixation or harvest of cells): About 14-17 days and 4 hours


SELECTION AGENT (mutation assays): 6-thioguanine
SPINDLE INHIBITOR (cytogenetic assays): no
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Triplicate in cytotoxicity assays; duplicate in mutation assays.

NUMBER OF CELLS EVALUATED: not specified but the colonies were counted by eye, excluding those with aproximately 50 cells or less.

DETERMINATION OF CYTOTOXICITY
- Method:
Relative Survival (%l) = (Average no. of colonies per treated culture/Average no. of colonies per vehicle control dish) x 100
Relative Population Growth (%) = (Treated culture population increase over the expression period/Vehicle control population increase over the expression period) x 100

OTHER EXAMINATIONS:
- Determination of polyploidy: not assessed
- Determination of endoreplication: not assessed
- Other: yes

Absolute Cloning Efficiency (ACE) (%) = (Average no. of viable colonies per dish/200) x 100
Mutant Frequency (%) = Total mutant clones/(no. of dishes x 2 x 10^5 x ACE)



Evaluation criteria:
The main criteria are presented here.

Assay acceptance criteria
An assay is considered acceptable if the following criteria are satisfied. The average absolute cloning efficiency of the vehicle controls should be between 70 and 15%. Assays with backgrounds mutant frequency higher than 15 x 10e-6 will not be used for test article evaluation. A positive control is included with each assay. The mutant frequencies for 5 treated cultures are normally determined in each assay.

Assay evaluation criteria
Mutation assays are performed with cell cultures exposed to 6 to 8 concentrations of test article. In addition to the confidence level to reach that is presented in section “Statistics” below, the mutant frequency must meet or exceed 15 x 10-6 in order to compensate for random fluctuations in the 0 to 10 X 10-6 background mutant frequencies that are typical for this assay. A test substance is mutagenic if a dose-related or toxicity-related increase in mutant frequency is observed (for at least 3 doses). A test article is nonmutagenic in a single assay if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to concentrations causing 10 to 15% survival or extends to a concentration at least 75% of that causing excessive toxicity.
Statistics:
The statistical tables provided by Kastenbaum and Bowman (1970) are used to determine whettaer the results at each dose level are
significantly different from the negative controls at 95% or 99% confidence levels. This test compares variables distributed according to Poissonian expectations by summing up the probabilities in the tails of two binomial distributions. The 95% confidence level must be met as one criterion for considering the test article to be active at a particular dose level.

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 nor precipitates, but tested up to recommended limit concentrations
Remarks:
(slight toxicity but not significant)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test artlcle did not alter the pH of the treatment medium outside the range of pH 7.0 to pH 7.8 at any applied concentration.
- Effects of osmolality: The test material had no effect on treatment medium osmolaJity over the range of concentrations used in the mutation assays. Osmolality measurements of treatment media from the nonactivation mutation assay showed the vehicl econtrol to be 263 mOsm/kg, the 2.5 mg/ml test article concentration to be 287 mOsm/kg and the high concentration (5.0 mg/ml) to be 319 mOsm/kg.
- Evaporation from medium: Not assessed
- Water solubility: Solubility testing with sterile deionized water showed that good solubility was maintained at 50.0 mg/ml
- Precipitation: The test article remained in solution in culture medium from O.0098 mg/ml to the maximumapplied concentration of 5.0 mg/ml.
- Other confounding effects: No

RANGE-FINDING/SCREENING STUDIES:
Ten doses levels were used in each case that ranged from 0.0098 to 5.0 mg/ml. The rangefinding cytotoxicity assay showed that the test material was nontoxic to CHO cells in cultures at all dose levels both with and without S9 metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: no

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Remarks on result:
other: strain/cell type: CHO-K1-BH4 cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Results in tabular format are provided in 'Attached background material'

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Methylal produced no toxicity and was considered negative for inducing forward mutations at the Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese Hamster Ovary (CHO) cells under the S9 metabolic activation and non activation conditions of the assay.
Executive summary:

The ability of test item, methylal, to induce forward mutations at Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese Hamster Ovary (CHO) cells under the S9 metabolic activation and non activation conditions of the assay was assessed.

A preliminary range-finding test showed that methylal was non toxic (with and without metabolic activation) for CHO cells at concentrations of 0.0098, 0.0195, 0.0391, 0.0781, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0 mg/ml. One negative (media) control and one vehicle control containing 10% sterile deionized water were used in each cytotoxicity. Triplicate cultures were used in cytotoxicity test. These results were used to select dose levels for the mutation assays with the concentration of 5.0 mg/ml accepted as upper limit for testing.

Mutation test was performed (with and without metabolic activation) at the concentrations of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 mg/mL. Two positive controls, 5-bromo-2-deoxyuridine without metabolic activation and 3-methylcholanthrene with metabolic activation and one negative (media) control were used in duplicate cultures. Methylal produced no toxicity and was considered negative for inducing forward mutations at the HGPRT locus in CHO cells under the S9 metabolic activation and non activation conditions of the assay.

Categories Display