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Toxicological information

Genetic toxicity: in vitro

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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:
other: Meets generally accepted scientific standards, well documented and 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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not applicable
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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:
other: TA 97a, TA 98, TA100, TA102
Additional strain 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:
- Vehicle(s)/solvent(s) used: ultrapure sterile water, DMSO
Controlsopen allclose all
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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.
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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
Negative controls:
yes
Remarks:
(= solvent controls)
Solvent 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 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
Species / strain:
other: TA 97a, TA 98, TA 100, TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Methoxyacetic acid concentration > 2 mg/plate
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
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.