Methoxyacetic acid

Genetic toxicity: in vitro

Administrative data

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Mutagenicity of ethylene glycol ethers and of their metabolites in Salmonella typhimurium his-

Author:

Hoflack JC, Lambolez L, Elias Z, Vasseur P

Year:

1995

Bibliographic source:

Mutat Res., 341: 281-287.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not applicable

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Methoxyacetic acid

EC Number:

210-894-6

EC Name:

Methoxyacetic acid

Cas Number:

625-45-6

Molecular formula:

C3H6O3

IUPAC Name:

2-methoxyacetic acid

Constituent 2

Reference substance name:

2-methoxyacetic acid

IUPAC Name:

2-methoxyacetic acid

Details on test material:

- Name of test material: Methoxyacetic acid (obtained from Merck, Darmstadt, Germany)
- Molecular weight: 90.08
- Analytical purity: > 99 %
- Physical state: liquid at ambient temperature
- Impurities: less than 0.005% peroxides and less than 0.001% aldehydes (data from manufacturer
- Purity: test compound was checked for purity by high definition GC/MS and NMR. Merck products contained less than
0.005% peroxides and less than 0.001% aldehydes (data from manufacturer).
- Expiration date: compounds were used for testing within 6 months after receipt or 1 month after organic synthesis.
- Storage condition: test material was stored in the dark at + 4°C in air-tight vials under nitrogen and in sterile conditions

Method

Target gene:

In the Salmonella typhimurium strains (TA 97a, TA 98, TA 100, TA102) the amino acid histidine locus is the target gene, in which induced back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+). The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between several base pair (TA 100, TA 102) and frameshift (TA 97a, TA 98) mutations.

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA100, TA102

Additional strain / cell type characteristics:

other: All strains have a defecient excision repair system (uvrB), which results in greatly enhanced sensitivity to some mutagens and considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (liver homogenate)

Test concentrations with justification for top dose:

Methoxyacetic acid was tested up to cytotoxic concentrations.
Up to 26 µmol/plate Methoxyacetic acid (2 mg/plate) were used.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ultrapure sterile water, DMSO

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with S9-mix (+ S9-mix)

Positive control substance:

benzo(a)pyrene

Remarks:

Positive control substance was dissolved in DMSO.

Migrated to IUCLID6: , 1 µg/plate, strain: TA 97a, TA 98, TA 100

Controls 2

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with S9-mix (+ S9-mix)

Positive control substance:

other: DHAC, 50 µg/plate, strain: TA 102

Remarks:

1,8-dihydroxyanthrachinon (DHAC) was dissolved in DMSO.

Controls 3

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with without S9-mix (- S9-mix)

Positive control substance:

9-aminoacridine

Remarks:

Positive control substance was dissolved in DMSO.

Migrated to IUCLID6: , 40 µg/plate, strain: TA 97a

Controls 4

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with without S9-mix (- S9-mix)

Positive control substance:

2-nitrofluorene

Remarks:

Positive control substance was dissolved in DMSO.

Migrated to IUCLID6: , 0.5 µg/plate, strain: TA 98

Controls 5

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with without S9-mix (- S9-mix)

Positive control substance:

sodium azide

Remarks:

Positive control substance was dissolved in ultrapure water.

Migrated to IUCLID6: , 2.5 µg/plate, strain: TA 100

Controls 6

Untreated negative controls:

yes

Remarks:

(= solvent controls)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Remarks:

, used in assay with without S9-mix (- S9-mix)

Positive control substance:

cumene hydroperoxide

Remarks:

Positive control substance was dissolved in DMSO.

Migrated to IUCLID6: , 75 µg/plate, strain: TA 102

Details on test system and experimental conditions:

METHOD OF APPLICATION: Overlay (soft) agar (plate incorporation method). The glucose concentration of base- and top-agars was 2%. Both agar types were used within 36 h after preparation to avoid dehydratation/increase of glucose concentration.

DURATION
- Preincubation period: No preincubation was performed. The dilutions of the compounds in ultrapure water were added to the top agar to an extent of 0.1 mL/plate, and the solutions of the chemicals diluted in DMSO to an extent of 0.2 mL/plate. After mixing of all components, soft agar was distributed directly on the plates.
- Exposure duration / Expression time / Selection time: Plates with microorganisms / test item situated in the topagar and controls were incubated at 37 °C and colonies were counted after 48 h incubation.

SELECTION AGENT (mutation assays): The selection is done by using a minimal agar that contains no histidine for the Salmonella typhimurium strains.

NUMBER OF REPLICATIONS: Three or four separate experiments were performed for each compound and each concentration was tested in triplicate plates.

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
- Doubling of the revertant colonies compared to solvent control
- Presence of a dose-effect relationship

Statistics:

No data given.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Methoxyacetic acid conc. > 2 mg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Methoxyacetic acid conc. > 2 mg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Methoxyacetic acid > 2 mg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Test results 4

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Methoxyacetic acid concentration > 2 mg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

In a reverse gene mutation assay in bacteria (Hoflack et al., 1995), strains TA 97a, TA 98, TA 100 and TA 102 of S. typhimurium were exposed to Methoxyacetic acid (> 99 % a.i.) at concentrations up to cytotoxicity of 2 mg/plate (vehicle: sterile water) in the presence and absence of mammalian metabolic activation (S9-mix) in the standard plate test (plate co-incubation). Methoxyacetic acid gave negative responses with every strain used, either with or without metabolic activation.