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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:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Publication

Data source

Reference
Reference Type:
publication
Title:
Evaluation of clastogenicity of formic acid, acetic acid and lactic acid on cultured mamalian cells.
Author:
Morita, T., Takeda, K. and Okumura, K.
Year:
1990
Bibliographic source:
Mutation Research 240: 195-202

Materials and methods

Principles of method if other than guideline:
Preparation of metabolic activation system
S9 (Oriental Yeast Co., Ltd., Japan) was derived from the livers of rats pretreated with pheno­barbital and 5,6-benzoflavone. The S9 activation system (S9 mix) was prepared just before use according to Matsuoka et al. (1979), and sterilized by filtration, except S9 fraction.
Chromosomal aberration test
Cells (2 x 104/dish) were cultured for 48 h before the test compounds were added. Each stock solution of formic acid, acetic acid or lactic acid was prepared in distilled water at 2 M and diluted appropriately with distilled water on the day of
use. An aliquot of 50-75 µl of diluted acid was added to the culture medium (5 ml). In the ab­sence of S9 mix, chromosome preparations were made by an air-drying method 24 h after the addition of each organic acid. Cytotoxicity of each acid was also examined by counting surviving cells. In the presence of S9 mix, the cells were washed with physiological saline after a 6-h treat­ment, and then incubated with fresh medium for 18 h before the chromosome preparations were made (Ishidate, 1987).
In order to study the effect of neutralization of the treatment medium, 2 kinds of treatment media were examined; one was adjusted to pH 5.8 or pH 6.0 with each of these acids and the other was so adjusted then immediately neutralized to pH 6.4 and pH 7.2 with 1 M NaOH. In order to study the effect of enhancement of the buffering ability, chromosomal aberration tests were carried out on these acids in the absence of S9 mix using F12 medium containing 34 mM NaHCO) (twice the concentration usually employed). Furthermore, to study the effect of alteration of the buffering system, the tests were performed on these acids in the absence of S9 mix using F12 medium contain­ing 30 mM HEPES instead of NaHC03 as a buffer. This medium was adjusted to pH 8.5 with NaOH, and the cells were incubated in closed culture vessels.
The pH of the medium was measured initially and at 6 hand 24 h after the treatment using a pH meter. 100 well-spread metaphases from 1 experi­ment were observed at each point to record the percentage (including gaps) of cells with chro­mosomal aberrations. At least 2 independent ex­periments were carried out in each case.
GLP compliance:
no
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
D,L-Iactic acid (CH)CHOHCO­OH, CAS No. 598-82-3, about 90% aqueous solution, pKa = 3.86) was purchased from Wako Pure Chemical Ind., Ltd. (Japan)

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The Chinese hamster ovary K1 cell line (CHO­Kl, Flow Laboratory, U.S.A.) was used. The cells were maintained in Ham's F12 medium (Flow) supplemented with 10% fetal calf serum, kanamy­cin (60 µg/ml) and 17 mM sodium bicarbonate (NaHC03). The cells were grown as monolayers at 370 C in a 5% CO2/95% air atmosphere. The cultures were regularly screened for mycoplasma contamination.
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate S9
Test concentrations with justification for top dose:
8-35 mM

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: 14-35 mM, when pH was <= 5.8

Any other information on results incl. tables

RS-Freetext:
When the culture medium was first acidified by the lactic
acid dose and neutralized to pH 6.4 or when medium is
containing 30 mM HEPES as buffer, lactic acid was
non-clastogenic.
Pseudo-positive reactions are seen as a result of
non-physiological low pH.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
other: results obtained need to be interpreted in the light of the medium pH

Using Chinese hamster ovary Kl cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHC03 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenio activity was observed. Using F12 medium supplemented with 34 mM NaHC03 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseUdo-positive reactions attributable to non-physio­logical pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.
Executive summary:

Using Chinese hamster ovary Kl cells, chromosomal aberration tests were carried out with formic acid, acetic acid and lactic acid, and the relationship between the pH of the medium and the clastogenic activity was examined. The medium used was Ham's F12 supplemented with 17 mM NaHC03 and 10% fetal calf serum. All of these acids induced chromosomal aberrations at the initial pH of ca. 6.0 or below (about 10-14 mM of each acid) both with and without S9 mix. Exposure of cells to about pH 5.7 or below (about 12-16 mM of each acid) was found to be toxic. When the culture medium was first acidified with each of these acids and then neutralized to pH 6.4 or pH 7.2 with NaOH, no clastogenio activity was observed. Using F12 medium supplemented with 34 mM NaHC03 as a buffer, no clastogenic activity was observed at doses up to 25 mM of these acids (initial pH 5.8-6.0). However, it was found that about 10% of the cells had aberrations at pH 5.7 or below (27.5-32.5 mM of each acid). Furthermore, when 30 mM HEPES was used as a buffer, chromosomal aberrations were not induced at doses up to 20 mM formic acid and acetic acid (initial pH 7.0-7.1), and at doses up to 30 mM lactic acid (initial pH 6.6). In the initial pH range of 6.4-6.7 (25-32.5 mM of each acid), chromosomal aberrations were observed. The above results show that these acids themselves are non-clastogenic, and the pseUdo-positive reactions attributable to non-physio­logical pH could be eliminated by either neutralization of the treatment medium or enhancement of the buffering ability.