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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:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restriction. The study was conducted basically in accord with the OECD 473 guideline "In Vitro Mammalian Chromosome Aberration Test". The only significant variation from this guideline was there were no positive controls reported. As the test materials produced positive results at acidic pH levels, the sensitivity of the procedure was demonstrated.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
positive control not included
GLP compliance:
not specified
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): formic acid
- Source: Wako Pure Chemical Ind., Ltd. (Japan)

Method

Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Additional strain characteristics:
other: substrain K1
Metabolic activation:
with and without
Metabolic activation system:
rat-liver S9 prepared from rats pretreated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
276, 368, 460, 552, 644, 920, 1150, 1266, and 138µg/mL (6 t0 30 mM)
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water
Controls
Negative controls:
yes
Positive controls:
no
Remarks:
positive control not required
Details on test system and conditions:
METHOD OF APPLICATION: instandard F12 medium


DURATION

- Exposure duration: 24 hours (with metabolic activation: cells were washed after 6 hours, and resuspended in fresh medium for another 18 hours)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours


SPINDLE INHIBITOR (cytogenetic assays): none. Fixation: air drying (according to reference: Ishidate, 1987)



NUMBER OF REPLICATIONS: 2 to 4


NUMBER OF CELLS EVALUATED: 100/experiment


DETERMINATION OF CYTOTOXICITY
- Method: surviving cell count


OTHER EXAMINATIONS:
- Determination of: chromatid gaps; chromosome gaps; chromatid breaks; chromosome breaks; chromatid exchanges; chromosome exchanges including dicentric and ring chromosomes


Evaluation criteria:
According to OECD Test Guideline no. 473
Statistics:
not reported

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
dependnent on pH (at pH 6 or below) and osmarity (cf. 3rd experiment)
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
not examined
Remarks on result:
other:
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Effects of pH:
- Toxicity: Exposure of cells to about pH 6.0 or below (12-14 mM) was toxic.
-Chromosome aberration: Formic acid induced chromosomal aberration at an initial pH of 6.1 - 6.3 (10-12 mM) in a dose-related manner. This effect was lower when the initial pH was adjusted to 6.4, and absent when the inittial pH was adjusted to 7.2 (cf. experiment 2)

- Effects of osmolality: toxicity and increased number of aberrant cells was seen at high osmolality (25 to 30 mM formic acid, F12 medium containing additional buffering substances at 30 to 34 mM buffer (cf. experiment 3).

Any other information on results incl. tables

In a series of experiments the influence of confounding factors, i.e. pH and osmolality) on the incidence of aberrant cells (%) was examined.

 

1) Incubation in standard F12 medium

Formic acid induced chromosomal aberration at an initial pH of >6.0 (10-12 mM) in a dose-related manner.

Exposure of cells to pH about 6.0 or less (12-14 mM formic acid) was cytotoxic.

 

Dose

pH

(at hours after start of incubation)

No. of cells scored

Aberrant cells (%)

Formic acid (mM)

Initial, 0 h

At 6 h

At 24 h

 

(-S9)

(+S9)

0

7.2

7.2

7.4

200

0

 

8

6.4

6.7

7.3

200

2.0

 

10

6.3

6.5

7.1

200

4.0

 

12

6.1

6.2

6.6

113

15.9

 

14

5.8

6.0

6.4

toxic

toxic

 

 

0

7.4

7.3

 

200

 

0

6

6.4

6.9

 

200

 

1.0

8

6.3

6.7

 

200

 

2.0

10

6.1

6.3

 

200

 

20.5

12

5.9

5.8

 

toxic

 

Toxic

 

2) Effect of neutralization of the medium

In a second set of experiments the initial pH of the medium was adjusted to pH 6.0 with 14 or 12 mM formic acid.

These media were then neutralized with 1 M NaOH to pH 6.4, and a second group to pH 7.2. These experiments were

also conducted with and without metabolic activation.

The results (table below; cell data were read from a graph [Figures 2 and 3 of the original publication] and are approximate)

indicate that the number of aberrant cells was not increased when the initial pH was appropriate, i.e. 7.2. The number of aberrant increased with decreasing pH-values.

 

 

Dose

pH

(hours after start of incubation)

Aberrant cells (%)

Formic acid (mM)

Initial, 0 h

Final, atAt24 h

(-S9)

(+S9)

12-14

6.0

6.8

12

 

 

6.4

7.2

4

 

 

7.2

7.3

0

 

 

12-14

6.0

6.4

 

33

 

6.4

7.1

 

2

 

7.2

7.2

 

3

 

3) Effect of buffer capacity

In a third set of studies, the effect of an increased buffer capacity was examined in the absence of metabolic activation. Two different buffer systems were used: i) the F12 medium containing 34 mM NaHCO3, and ii) F12 medium containing 30 mM HEPES.

Under these conditions, there was no clastogenic activity of formic acid up to 20 or 25 mM, the initial pH being in the range 7.1 to 7.4.

Depending on the buffer used, aberrant cells were seen at 25 or 27.5 mM and above. At 30 mM the formic acid was cytotoxic irrespective of the buffer system. Acidic pH levels were seen in the medium containing 34 mM NaHCO3at 25 mM and above, and at 30 mM in the medium containing 30 mM HEPES, i.e. the buffer capacity was exhausted and the pH was low.

Overall, low pH and increased ionic strength caused cytotoxicity and an increase in aberrant cells.

 

Dose

pH

(hours after start of incubation)

No. of cells scored

Aberrant cells (%)

Formic acid (mM)

Initial, 0 h

At 6 h

At 24 h

 

NaHCO3
34 mM

HEPES
30 mM

0

7.4

7.3

7.4

400

0

 

20

6.1

7.1

7.3

200

0.5

 

25

5.8

6.8

7.1

400

0.5

 

27.5

5.7

6.5

6.7

200

10.5

 

30

5.4

6.4

6.7

toxic

15.9

 

 

 

 

 

 

 

 

0

8.5

7.9

7.4

400

 

0

10

7.6

7.3

6.9

200

 

0.5

20

7.1

7.1

6.8

200

 

0

25

6.7

6.6

6.4

200

 

12

30

5.9

5.9

5.9

toxic

 

toxic

 

Applicant's summary and conclusion

Conclusions:
CL-Freetext:
It was concluded that formic acid is not itself clastogenic to these cells but that the acidic conditions were responsible for the chromosome aberrations observed.

Likewise, the cytotoxicity depended on the pH value when formic acid was tested up to cytotoxic concentrations.
Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration, conducted similar to OECD Test Guideline No. 473) CHO cell cultures were exposed to formic acid dissolved in F12 cell culture medium at concentrations of 6 to 14 mM, i.e. 0, 276, 368, 460, 552, and 644 µg/mL with and without metabolic activation. In a series of subsequent experiments the influence of confounding factors, i.e. pH and osmolality) on the incidence of aberrant cells (%) was examined at concentrations of 20, 25, 27.5, and 30 mM, i.e. at 920, 1150, 1266, and 1380 µg/mL.

 

Formic acid was tested up to cytotoxic concentrations. Overt cytotoxicity and increased numbers of aberrant cells were seen when the initial pH of the incubation medium was approximately 6 or less. The number of aberrant cells was not increased by formic acid up to 14 mM, i.e. 644 mg/mL, if the initial pH was adequate (pH 7.2). Moreover, no positive response was seen with concentrations up to 20 mM (920 µg/mL) with two different buffer systems as long as the buffer capacity was not exhausted. At 25 to 30 mM formic acid an increasing positive response and cytotoxicity were both seen. It was concluded that this results from the combined inadequately low pH and high osmolarity of the incubation medium.

  

Positive controls were not included. Acetic and lactic acid were included and showed similar results. There was no evidence of Chromosome aberration induced over background by formic, acetic or lactic acid themselves. Pseudo-positive reactions attributable to non-physiological pH could be eliminated by either neutralisation of the treatment medium or enhancing the buffer capacity.

 

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 473 in  Chinese Hamster ovary cells for in vitro cytogenetic mutagenicity data. 

Conclusion:

Formic acid itself is not clastogenic. A pseudo-positive response was attributable to non-physiologically low pH.