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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other:
Remarks:
The study is performed in line with OECD guideline 473 (in vitro mammalian chromosomal aberration test), materials and methods are well described. A deviation from this guideline is that the test is only performed without metabolic activation. Additionally, there are no detailed information on the results.

Data source

Reference
Reference Type:
publication
Title:
Primary mutagenicity screening of food additives
Author:
Ishidate Jr., M. Sofuni, T., Yoshikawa, K., Hayashi, M., Nohmi, T., Sawada, M., Matsuoka, A.
Year:
1984
Bibliographic source:
Food Chemistry and Toxicology, Vol. 22, No. 8, p 623-636

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Version / remarks:
Tested without metabolic activation
Principles of method if other than guideline:
- Principle of test: Chromosomal aberration tests were carried out using a Chinese hamster fibroblast cell line, CHL.The cells were exposed to each sample at three different doses for 24 and 48 hr. A hundred well-spread metaphases were observed under the microscope (x 600 with a no- cover objective lens).
- Short description of test conditions: The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to each sample at three different doses for 24 and 48 hr. In the present studies, no metabolic activation systems were applied. The maximum dose of each sample was selected by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd). Chromosome preparations were made as follows. Colcemid (final concn 0.2µg/mL) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a no- cover objective lens).
- Parameters analysed / observed: The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/mL) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/mL).
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Reference substance name:
ethandioic acid
Cas Number:
144-62-7
Molecular formula:
C2H2O4
IUPAC Name:
ethandioic acid
Test material form:
not specified

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese hamster lung fibroblasts (V79)
- Suitability of cells:

For cell lines:
- Number of passages if applicable: n/a
- Methods for maintenance in cell culture: The cells had been maintained by 4-day passages and grown in a monolayer in petri dishes with Eagle's MEM (GIBCO) supplemented with 10% calf serum.
- Cell cycle length, doubling time or proliferation index : doubling time approximately 15 h
- Modal number of chromosomes: 25

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Eagle's MEM (GIBCO) supplemented with 10% calf serum, other conditions not reported.
Metabolic activation:
without
Test concentrations with justification for top dose:
Three different doses were tested. The maximum dose (0.125 mg/mL) was determined by a preliminary test in which the dose needed for 50% cell-growth inhibition was estimated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiol. saline

- Justification for choice of solvent/vehicle: not reported

Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
not specified
Remarks:
Incidence of aberrations in the negative control < 3%
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: The cells were exposed to each sample at three different doses for 24 and 48 hr.
- Number of independent experiments : not reported

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 and 48h

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colcemid; 0.2 µg/mL
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A hundred well-spread metaphases were observed under the microscope (x 600 with a no- cover objective lens).
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/mL) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/mL).


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: not reported

Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. When no reasonable dose-response relationships were found, additional experiments were carried out at similar dose levels. For a quantitative evaluation of the clastogenic potential of the positive samples, the D20 was calculated, which is the dose (mg/mL) at which structural aberrations (including gaps) were detected in 20% of the metaphases observed. In addition, the TR value was calculated, which indicates the frequency of cells with exchange-type aberrations per unit dose (mg/mL).

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
without
Genotoxicity:
negative
Remarks:
0% structural aberrations and only 4% polyploidy after 48h
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not examined
Positive controls validity:
not specified

Any other information on results incl. tables

Table 1: Results of chromosomal aberrations in vitro

Substance

Max dose (mg/mL)

Solvent

Polyploid (%)

Structural aberrations

Result

(%)

(h)

Oxalic acid

10.0

Saline

4

0.0

48

negative

Applicant's summary and conclusion

Conclusions:
In the present publication of Ishidate et al. 1984 several food additives incl. Oxalic acid were evaluated for their mutagenic potential as well as in the chromosome aberration assay. CHL cells were incubated with three different concentrations of the respective food additive for 24 and 48h. Afterwards they were treated with colcemid and harvested. The cells were fixed with acetic acid-methanol, spread on glass slides and stained with Giemsa. Hundred well.spread metaphases were evaluated and considered positive if the incidence of aberrations was more than 10% and negative if the incidence was less than 4.9%. Polyploidy was found with an incidence of 4% in the highest dose tested whereas no structural aberrations were found after treatment with Oxalic acid for 48h. Thus, Oxalic acid is not considered to be a genotoxic substance under the conditions of the test.
Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 476, Chinese hamster lung fibroblasts (V79) cultured in vitro were exposed to oxalic acid in physiological saline in different concentrations in the absence of mammalian metabolic activation (S9 mix) up to 10 mg/mL for 24 and 48 h.


The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.


This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.