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Genetic toxicity: in vitro

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Administrative data

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.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Formic acid
EC Number:
200-579-1
EC Name:
Formic acid
Cas Number:
64-18-6
Molecular formula:
CH2O2
IUPAC Name:
formic acid
Details on test material:
- 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
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

Method

Target gene:
HPRT locus
Species / strain
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
Controls
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 .

Results and discussion

Test results
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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative

Sodium formate is considered to be negative. 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.
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) Sodium 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.

 

 

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