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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
GLP guideline study. Suitability of the test substance: formic acid is almost exclusively present as formate anoin in aqueous solution at neutral pH. Data on formic acid may therefore be used to assess formate salts.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): formic acid
- Purity test date: formic acid 85.3%, water 14.3%.
- Composition of test material, percentage of components: formic acid 85.3%, water 14.3%.
- Stability under test conditions: yes
- Storage condition of test material: room temperature
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- culture medium: Ham's F12 mediumwith glutamine and hypoxanthine supplemented with fetal calf serum
- pretreatment mediu: culture medium with HAT (hypoxanthine, aminopterin, thymidine)
- selection medium: glutamine- and FCS-supplemented, hypoxanthine-free Ham's F12 medium with 6-thioguanine
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not required. Stocks of the CHO cell line (1 -ml portions) were maintained at -196°C in liquid nitrogen.
Additional strain / cell type characteristics:
other: Substrain K1: high proliferation rate (doubling time of about 12 - 16 hours); high plating efficiency (about 90% ); karyotype with a modal number of 20 chromosomes .
Metabolic activation:
with and without
Metabolic activation system:
without/with S-9 mix from Aroclor 1254 treated male Sprague-Dawley rats
Test concentrations with justification for top dose:
Without S9: 0, 31.25, 62.5, 125, 250, and 500 μg/mL
With S9: 0, 25, 50, 100, 200, and 400 μg/mL
Vehicle / solvent:
aqueous culture medium, Ham's F12
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate, methylcholanthrene
Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): one week
- Fixation time: approx. 15 days


SELECTION AGENT (mutation assays): 6-thioguanine


NUMBER OF REPLICATIONS: 6
NUMBER OF EXPERIMENTS: 2


NUMBER OF CELLS EVALUATED: all colonies were counted


DETERMINATION OF CYTOTOXICITY
- determined in a pretest
- concentration range: 0.1-500 µg/mL
- exposure period: 4 hours
NUMBER OF REPLICATIONS: 2
NUMBER OF EXPERIMENTS: 2

- Method: cloning efficienc (survival, viability)


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
- Cell morphology was checked in cultures of all test groups after 3 hours of treatment.
- The pH value and osmalality were measured.
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


Evaluation criteria:
Colonies of each test group were fixed, Giemsa stained and counted.
Mutant frequency was calculated from the uncorrected mutant frequency divided with cloning efficiency (viability).
Criteria for positive response:
Increases of the corrected mutation frequencies both above the concurrent negative control values and the historical negative control range.
Evidence of reproducibility of any increase in mutant frequencies.
A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship .
Statistics:
Due to the negative findings, a statistical evaluation was not carried out .
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: approx. >300-500 g/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mutant frequency
Formic acid: there was no increase in the number of mutant colonies  observed either with or without metabolic activation.
Controls: negative and positive controls gave results as expected.

Cytotoxicity
Without S9: number of colonies and cell density were not reduced at 500  µg/mL.
With S9: cytotoxicity noted from 200-400 µg/mL onward only in the 2nd  experiment.

Morphology
Cell attachment was reduced only in the 2nd experiment with S9 from about  400 µ/mL onwards.

PH-values
The pH was 5.5 and 6.5, i.e. pH was influenced in the 2nd experiment at  500 µg/mL.
Remarks on result:
other: other: HPRT
Remarks:
Migrated from field 'Test system'.

1-    Without metabolic acitvation

 

Test groups

doses

Mutant frequency (per 106 cells)

(corrected; taking into account absolute cloning

efficiency 2 at the end of the expression period)

without metabolic activation

 

1stexperiment

2ndexperiment

Vehicle control

2.96

2.88

31.25 µg/mL

1.31

 

62.5 µg/mL

1.26

 

100 µg/mL

 

2.89

125 µg/mL

1.32

 

200 µg/mL

 

8.80

250 µg/mL

1.50

 

300 µg/mL

 

1.33

400 µg/mL

 

5.33

500 µg/mL

2.44

3.06

300 µg/mL EMS

295.88

302.03

EMS =ethyl methane sulfonate

 

 

2-    With metabolic acitvation

 

 

Test groups

doses

Mutant frequency (per 106 cells)

(corrected; taking into account absolute cloning

efficiency 2 at the end of the expression period)

with metabolic activation

 

1stexperiment

2ndexperiment

Vehicle control

4.05

3.54

25 µg/mL

1.87

 

50 µg/mL

1.57

 

100 µg/mL

2.79

1.87

200 µg/mL

2.83

0.94

300 µg/mL

 

5.18

400 µg/mL

6.07

0.00

(toxicity)

500 µg/mL

 

-

(discontinued due to severe toxicity)

10 µg/mL MCA

242.94

149.02

MCA = methylcholanthrene

Conclusions:
Interpretation of results (migrated information):
negative

Potaasium formate is considered to be negative. Reason: negative data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.
Executive summary:

In a mammalian cell gene mutation assay (HPRT locus), Chinese Hamster ovary cells cultured in vitro were exposed to formic acid (85.3%) at concentrations of 0, 31.25, 62.5, 125, 250, and 500 μg/mL in the presence, and of 0, 25, 50, 100, 200, and 400μg/mL in the absence of mammalian metabolic activation. 

 

Formic acid was tested up to cytotoxic concentrations (i.e., 200 to 400 µg/mL in the absence, and 400 to 500 µg/mL in the presence of metabolic activation) without increasing mutation frequency at any concentration.  The positive controls did induce the appropriate response as did the vehicle control. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable because it meets the requirements of GLP and current test guidelines.  This study satisfies the requirement for Test Guideline OECD 476 and EEC Directive 2000/32, B.17 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Conclusions:

1) Formic acid did not induce forward mutations in vitro in the CHO/HPRT assay, with or without metabolic activation.

2) Potassium formate is not mutagenic in mammalian cells. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

 

 
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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
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. Suitability of the test substance: formic acid is almost exclusively present as formate anoin in aqueous solution at neutral pH. Data on formic acid may therefore be used to assess formate salts.
Justification for type of information:
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. Suitability of the test substance: formic acid is almost exclusively present as formate anoin in aqueous solution at neutral pH. Data on formic acid may therefore be used to assess formate salts.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
positive control not included
GLP compliance:
not specified
Type of assay:
other: in vitro chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): formic acid
- Source: Wako Pure Chemical Ind., Ltd. (Japan)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type 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 to 30 mM)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
no
Remarks:
positive control not required
Details on test system and experimental 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
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1 cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dependnent on pH (at pH 6 or below) and osmarity (cf. 3rd experiment)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not examined
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).

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

 

Conclusions:
Interpretation of results (migrated information):
negative

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. Potassium formate is equally negative. Data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

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 (Morita, 1990).

 

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. 

Conclusions:

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

2) The same applies to potassium formate. Data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Suitability of the test substance: formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH. Data on formic acid may therefore be used to assess formate salts.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): formic acid
- Analytical purity: 97.4%
Target gene:
reversion in the His-operon
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Metabolic activation:
with and without
Metabolic activation system:
10% and 30% induced male Sprague Dawley rat liver S9 and induced male Syrian hamster liver S9
Test concentrations with justification for top dose:
0, 10, 33, 100, 333, 1000, 3333 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-phenylenediamine in strains TA98 in the absence of S-9; 2-aminoanthracene in all strains in the presence of S-9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation


DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours



SELECTION AGENT (mutation assays): histidine



NUMBER OF REPLICATIONS: chemicals were tested in triplicate; repeat experiments were conducted


NUMBER OF CELLS EVALUATED: his+ revertants


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


Evaluation criteria:
A chemical was judged to be mutagenic if it produced a reproducible, dose-related response over the solvent control, under a single metabolic activation condition, in replicate trials. According to OECD TG 471.
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

-       Strain TA1535

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

12

0.7

26

2.4

9

1.7

8

2.5

11

1

18

1.2

10     

 

 

21

2.7

11

1.3

 

 

13

2.6

 

 

33     

16

1.7

15

0.9

10

0.9

9

2.3

16

0.7

20

4.3

100     

13

0.9

19

1.2

11

1.2

12

2.6

14

2.6

15

0.9

333     

8

0.7

13

0.3

11

1.8

12

1.7

10

1

16

0.6

1000     

13

2.3

13

0.6

12s

1.2

7s

1.2

10

2.1

7s

0.7

3333     

11

1.7

 

 

 

 

4s

2.1

 

 

3s

1.5

Positive Control

506

24.1

332

10.2

180

18.4

340

22.8

214

27.7

60

2.3

      

     - Strain: TA100

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

133

2.7

151

11

171

3.2

159

9.2

162

5.1

162

9.1

10     

 

 

164

8.4

175

3.8

 

 

157

12.2

 

 

33     

132

7.1

152

10.1

160

9.8

154

4.3

162

9.6

170

3.1

100     

131

3.5

157

13.3

161

5.8

139

3.6

167

5.7

161

9.5

333     

138

0.3

153

9.8

175

5.2

138

7.5

170

8.5

151

9

1000     

137

6.1

125

15.3

165

15

111

3.5

157

7.9

112

10.7

3333     

68s

9.7

 

 

 

 

81s

4.6

 

 

46s

20.2

Positive Control

319

10.5

316

3.5

657

5.1

399

4.9

599

23.1

447

17.5

      

     - Strain: TA97

Dose

No Activation
(Equivocal)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Equivocal)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

144

9.3

175

14.6

207

11.1

172

4.1

204

19.6

213

5.9

178

15.4

10     

 

 

181

7.1

213

9.4

 

 

 

 

211c

9.5

 

 

33     

179

9.8

182

9.2

214

9.7

150

6.6

214

7.8

214

3.5

191

9.4

100     

179

6

180

10.5

206

1.8

167

34

213

12.5

201

24.8

182

4.4

333     

165

8.1

179

9.5

222

2.1

213

13

196

16.2

203

10.4

188

17.6

1000     

122

2.7

185

11.9

170

8.6

146

14.6

158s

10.7

153

5.2

109

7.2

3333     

65

6.1

 

 

 

 

36s

10.2

65s

6.7

 

 

85s

17.1

Positive Control

415

11.2

403

11.1

463

15.8

368

24.9

451

19.6

449

10.1

454

25.6

      

     -Strain: TA98

Dose

No Activation
(Negative)

No Activation
(Negative)

10% HLI
(Negative)

30% HLI
(Negative)

10% RLI
(Negative)

30% RLI
(Negative)

Protocol

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

Preincubation

ug/Plate

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

Mean

± SEM

0     

24

2.6

24

0.3

42

2.1

32

0.3

36

3.7

40

1.5

10     

 

 

21

2.3

18

0.6

 

 

28

5.2

 

 

33     

24

2

17

1

22

2.8

31

1.2

33

5.4

35

4

100     

24

1.8

20

2

42

9.4

26

0.3

30

4.8

31

1.5

333     

21

3.5

14

0.3

33

4

26

0.6

23

1.5

23

2.3

1000     

21

2.3

20

2

35

2.2

25

1.3

24

3.5

25

2

3333     

18s

0

 

 

 

 

11

1.5

 

 

7s

4

Positive Control

380

5.6

423

27.7

202

16

147

9.3

202

10.9

83

7.3

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity

 

Conclusions:
Interpretation of results (migrated information):
negative

Potassium formate is considered to be negative. Reason: negative data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.
Executive summary:

Formic acid was tested in an in-vitro genotoxicity test using bacteria (TA97, TA98, TA100, and TA1535) with and without metabolic activation (supernatant from induced male rat and Syrian hamster liver) at concentrations of 0, 10, 33, 100, 333, 1000, and 3333 µg/plate in accordance with OECD Guideline No. 471. Solvent and positive controls were included and performed as expected. Tests were conducted in triplicate, and two independent experiments were conducted. The number of revertants was not increased in any strain with or without metabolic activation up to and including the top dose of formic acid. Bacteriotoxicity was seen at 1000 µg/plate and above (Zeiger, 1992).

 

Conclusions:

1) Formic acid lacked genotoxicity in a valid bacterial cell in-vitro test performed according to OECD Guideline No. 471.

2) Potassium formate is not mutagenic in bacteria. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
Version / remarks:
Exceeds guideline study in that the effect of the pH value was clearly elucidated
GLP compliance:
no
Type of assay:
Drosophila SLRL assay
Specific details on test material used for the study:
- Name of test materials (as cited in study report): Formic acid
Sodium formate; produced by neutralization of 0.1% formic acid with glycine-NaOH buffer (feeding experiment)
Species:
Drosophila melanogaster
Strain:
other: Oregon-K
Sex:
male
Details on test animals or test system and environmental conditions:
Environment: 24°C
Route of administration:
oral: feed
Vehicle:
- Vehicle(s)/solvent(s) used: feed was used to allow dosing of both the liquid acid and the solid sodium formate salt
Details on exposure:
Oregon-K strain of Drosophila melanogaster were treated using dosed feed with 0.1 % formic acid, or sodium formate produced by neutralization of 0.1% formic acid with glycine-NaOH buffer.
Vapor phase exposure: Males were exposed 24 h in specially designed bottles containing 5 ml of the test substance. The flies themselves were kept in small glass tubes which were closed with gauze on the lower end and with cotton wool on the upper end.
Larval feeding experiment: A nutrient containing agar, brewer's yeast, corn meal, sucrose and water was prepared, autoclaved, cooled and supplemented with the chemicals to be tested. Females were allowed to lay eggs for 24 to 48 h and were then discarded. Males, which spent their whole larval life on the medium, were collected 24 h after emergence and mated individually to 2 females. The pH of the feed varied between 4.2 to 5.6, depending on the added reagent.
Matings: the Muller-5 technique was used to test for sex-linked lethals. About 50 males were individually crossed with one or two virgins. Every three days the males were transferred to fresh virgins in order to produce three successive broods.
Duration of treatment / exposure:
entire larval stage
Dose / conc.:
0.1 other: % as formic acid
No. of animals per sex per dose:
About 50 treated males were mated with M-5 virgins and every third day the males were transferred to two fresh virgins in order to produce three successive broods
Statistics:
Statistical evaluation included the chi-square test, the rank correlation method of Kendall (1955), or a special test for mutation frequency of Stevens (1942).
Sex:
male
Genotoxicity:
negative


1) Vapor experiment
Exposure to 0.1% Formic Acid (FA) significantly (p<0.001) increased the  

number of mutations compared to controls.
=========================================================
                               Brood
           1st            2nd          3rd       total
---------------------------------------------------------
         n     %        n     %      n     %      n     % 
---------------------------------------------------------
FA     1068  1.12     1130  1.42    850  1.41   3048  1.31 

Historical control                              2584  0.15
=========================================================
n = number of chromosomes tested
% = percentage of sex-linked lethals found     
                                               

2) Larval feeding experiments
Exposure to 0.1% Formic Acid (FA) increased the number of mutations 

without gaining statistical significance between broods or compared to 

controls.
===========================================================
                               Brood
           1st            2nd          3rd       total
-----------------------------------------------------------
         n     %        n     %      n     %      n     % 
-----------------------------------------------------------
FA      786  1.15      522  1.34    671   0.88   1879  1.11
NaF                    544  0.38

Historical control                               2584  0.15
===========================================================
n = number of chromosomes tested
% = percentage of sex-linked lethals found 
NaF = Formic acid, neutralized to pH = 7.5 using a 0.1 M glycine-NaOH buffer.                                                  

The sodium formate (NaF; produced from formic acid by neutralization) sex-linked lethal was not different from the control. 

Formic acid results of the feeding study were also stated as not being significantly different from control.

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

Formic acid was tested for genetic toxicity in a multigenerational test in Drosophila melanogaster similar to the OECD Guideline No. 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster). Following exposure to 0.1% formic acid vapour, the number of mutants was significantly increased compared to historical controls (p<0.001).

An increase was also seen with 0.1% formic acid in a subsequent feeding experiment, but without gaining statistical significance.  Sodium formate (produced by neutralization of formic acid) at the same molar concentration in the feed was negative in the Drosophila SLRL test. The authors concluded that the mutations observed with formic acid were related to the acidic pH, rather than to the acid or the formate molecule itself (Stumm-Tegethoff, 1969).

Conclusion:

Formic acid and sodium formate did not induce mutations in the Drosophila SLRL test in vivo.

This result can be read across to other formate salts including potassium formate.

 

 

 

 

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

No studies with potassium formate are known to exist, but results on other formate salts or formic acid may be extrapolated as justified in section 7.1.1.

Genetic toxicity in vitro

Mutagenicity in bacteria:

Formic acid was tested in an in-vitro genotoxicity test using bacteria (TA97, TA98, TA100, and TA1535) with and without metabolic activation (supernatant from induced male rat and Syrian hamster liver) at concentrations of 0, 10, 33, 100, 333, 1000, and 3333 µg/plate in accordance with OECD Guideline No. 471. Solvent and positive controls were included and performed as expected. Tests were conducted in triplicate, and two independent experiments were conducted.The number of revertants was not increased in any strain with or without metabolic activation up to and including the top dose of formic acid.Bacteriotoxicitywas seen at 1000 µg/plate and above (Zeiger, 1992).

 

Conclusions:

1) Formic acid lacked genotoxicity in a valid bacterial cell in-vitro test performed according to OECD Guideline No. 471.

2) Potassium formate is not mutagenic in bacteria. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Mutagenicity in mammalian cells:

In a mammalian cell gene mutation assay (HPRT locus), Chinese Hamster ovary cells cultured in vitro were exposed to formic acid (85.3%) at concentrations of 0, 31.25, 62.5, 125, 250, and 500μg/mL in the presence, and of0, 25, 50, 100, 200, and 400μg/mL in theabsence of mammalian metabolic activation. 

 

Formic acidwas tested up to cytotoxic concentrations (i.e., 200 to 400 µg/mL in the absence, and 400 to 500 µg/mL in the presence of metabolic activation) without increasing mutation frequency at any concentration.  The positive controls did induce the appropriate response as did the vehicle control. There was no evidence of induced mutant colonies over background.

 

This study is classified as acceptable because it meets the requirements of GLP and current test guidelines.  This study satisfies the requirement for Test Guideline OECD 476 and EEC Directive 2000/32, B.17 for in vitro mutagenicity (mammalian forward gene mutation) data.

 

Conclusions:

1) Formic acid did not induce forward mutations in vitro in the CHO/HPRT assay, with or without metabolic activation.

2) Potassium formate is not mutagenic in mammalian cells. Reason: data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Chromosome aberration

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 (Morita, 1990).

 

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. 

Conclusions:

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

2) The same applies to potassium formate. Data on formic acid may be used to assess formate salts, because formic acid is almost exclusively present as formate anion in aqueous solution at neutral pH.

Genetic toxicity in vivo

Formic acid and sodium formate were both tested for genetic toxicity in a multigenerational test in Drosophila melanogaster similar to the OECD Guideline No. 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster). Following exposure to 0.1% formic acid vapour, the number of mutants was significantly increased compared to historical controls (p<0.001).

An increase was also seen with 0.1% formic acid in a subsequent feeding experiment, but without gaining statistical significance.  Sodium formate(produced by neutralization of formic acid) at the same molar concentration in the feed was negative in the Drosophila SLRL test. The authors concluded that the mutations observed with formic acid were related to the acidic pH, rather than to the acid or the formate molecule itself (Stumm-Tegethoff, 1969).

Conclusion:

Formic acid and sodium formate did not induce mutations in the Drosophila SLRL test in vivo.

This result can be read across to other formate salts including calcium diformate.

 



Short description of key information:
Sodium formate and formic acid lack genotoxic properties. This can be extrapolated to other formate salts including potassium formate.
This is also supported by the finding that another formate salt, potassium diformate, lacked oncogenicity in rats and mice (cf. respective section).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification. Criteria of regulations 67/548/EC and 1272/2008/EC are not met.