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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(May 19, 2000)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Glyoxal
EC Number:
203-474-9
EC Name:
Glyoxal
Cas Number:
107-22-2
Molecular formula:
C2H2O2
IUPAC Name:
oxalaldehyde
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: B 61
- Purity test date: October 31, 2001

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Glyoxal as 40% aqueous solution is stable for 6 months. The stability of the test substance as such and throughout the study period had been verified by reanalysis
- Solubility and stability of the test substance in the solvent/vehicle: the stability of the test substance (aqueous solution) at room temperature in the vehicle water also had been determined analytically in the course of the determination of the reanalysis

Method

Target gene:
All strains are mutants from histidine auxotrophy (his) to histidine prototrophy (his+)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: defect in the excision repair system (uvrB)
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: AT base pair at the primary reversion site
Metabolic activation:
with and without
Metabolic activation system:
S-9 fraction from the liver of male Sprague-Dawley rats treated with AROCLOR 1254 mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer)
Test concentrations with justification for top dose:
- Test concentrations in the first experiment (TA 1535, TA 100, TA 102, TA 1537, TA 98): 0, 52, 260, 1300, 6500 and 13000 µg/plate
- Test concentrations in the second experiment (TA 100, TA 102): 0, 1000, 2000, 3000, 4000, 5000 µg/plate
- The concentrations tested refer to the aqueous test substance and not to the active ingredient

Vehicle / solvent:
Due to the good solubility of the test substance in water, water was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
(without S9 mix)
Positive control substance:
other: N-methyl-N´-nitro-N-nitrosoguanidine (MNNG)
Remarks:
(in DMSO at 5 µg/plate, in TA 1535 and TA 100)
Positive controls:
yes
Remarks:
(without S9 mix)
Positive control substance:
other: 4-nitro-o-phenylendiamine (NOPD)
Remarks:
(in DMSO at 10 µg/plate; in TA 98)
Positive controls:
yes
Remarks:
(without S9 mix)
Positive control substance:
9-aminoacridine
Remarks:
(in DMSO at 100 µg/plate; in TA 1537) Migrated to IUCLID6: (AAC)
Positive controls:
yes
Remarks:
(without S9 mix)
Positive control substance:
mitomycin C
Remarks:
(in DMSO at 0.25 µg/plate; in TA 102) Migrated to IUCLID6: (Mit. C)
Positive controls:
yes
Remarks:
(with S9 mix)
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
in DMSO at 2.5 µg/plate in TA 1535, TA 100, TA 1537, TA 98; in DMSO at 3.0 µg/plate in TA 102
Details on test system and experimental conditions:
Standard plate test (plate incorporation method, based on Ames et al., Mut. Res., 31, 347-364, 1975 and Proc. Nat. Acad. Sci. USA, 70, 782-786, 1973):
- Soft agar was prepared from 100 mL agar (0.6% agar + 0.6% NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants consisting of 0.5 mM histidine and 0.5 mM biotin);
- Two ml portions of the soft agar were transferred into test tubes, which were then kept in a water bath at 45 °C;
- In each tube, 0.1 mL test solution or vehicle, 0.1 mL fresh bacterial culture and 0.5 mL of either S9 mix or phosphate buffer were added;
- The content of each tube was mixed and poured onto Vogel-Bonner agar plates (minimal glucose agar plates);
- The plates were incubated at 37 °C for 48 to 72 hours;
- Three test plates per test concentration or control were used;
- At the end of the incubation time, the bacterial colonies (his+ revertants) were counted;
- Cytotoxicity was determined in all test groups and both experiments, with and without S-9 mix; the plates were examined for decrease in the number of revertants, diminution of background lawn, and reduction in titer;
- The titer was determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the 2 highest doses in both experiments;
- Precipitation of the test material was recorded.
Evaluation criteria:
The test chemical is considered positive if the following criteria is met:
Dose-related and reproducible increase in the number of revertant colonies, i .e. about doubling of the spontaneous mutation rate in at least one tester strain either with or without S-9 mix;
The test chemical is considered negative if the following criteria is met:
The number of revertants for all tester strains is within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
The mean number of revertant colonies per plate and the standard deviations were determined for all dose groups as well as for the positive and negative (vehicle) controls in both experiments.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
Experiment 1: an increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.3) and at 6500 µg/plate (factor 2.2). Experiment 2: an increase in the number of mutant colonies was observed at 2000 and 3000 µg/plate
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Experiment 1: an increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.2). Experiment 2: an increase in the number of mutant colonies was observed from 1000 µg/plate (factor 2.8) onwards.
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Experiment 1: a slight increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.0). Experiment 2: a slight increase in the number of mutant colonies was seen in the second experiment at 2000 µg/plate (factor 1.6).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
Experiment 1: a slight increase in the number of mutant colonies was observed at 1300 µg/plate (factor 2.0). Experiment 2: a slight increase in the number of mutant colonies was observed at 2000 µg/plate (factor 1.7).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of mutant colonies was observed (factors ranging from 0.1 to 1.3 in absence of metabolic activation and from 0.3 to 1.7 in presence of metabolic activation).
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed depending on the tester strain and test conditions from 4000 to 6500 µg/plate onward.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid

Applicant's summary and conclusion