Registration Dossier

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test substance: 2-ethylhexyl-cyanoacetate
- Substance number: 96/219
- Batch number: ABL. NR. 13-9844
- CAS number: 13361-34-7
- Degree of purity/content: 98.5 g/100 g (GC)
- Date of manufacturing: 27-DEC-1995
- Physical state/appearance: liquid, colorless to light yellowish
- Stability under test conditions: The stability of the TS over the study period has been proven by reanalysis. The stability of the TS in the vehicle DMSO and in water was assured analytically.
- Storage conditions: room temperature

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
Standard plate test: 20, 100, 500, 2500 and 5000 µg/plate
Preincubation test: 20, 100, 500, 1000 and 2000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
With S-9: 2.5 µg 2-AA (TA 100, TA 98, TA 1537, TA 1535); 60 µg 2-AA (E. coli WP2 uvrA). Without S-9: 5 µg MNNG (TA 100, TA 1535), 10 µg NOPD (TA 98), 100 µg AAC (TA 1537), 10 µg ENNG (E. coli WP2 uvrA).
Details on test system and experimental conditions:
STANDARD PLATE TEST (SPT): 3 test plates per dose or per control
1. Salmonella typhimurium:
The experimental procedure is based on the method of Ames et al. Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM
histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar: 980 ml aqua dest., 20 ml Vogel-Bonner E medium, 15 g Difco bacto agar, 20 g D-glucose, monohydrate.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

2. Escherichia coli:
The experimental procedure is based on the method of Ames et al. Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6% agar + 0.6% NaCl) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at 45°C and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SAl selective agar) is based on the description of Green, M.H.L. and Muriel, W.J., with the exception of solution E (tryptophan solution), which has been added to the soft agar before: 300 ml solution B (agar), 100 ml solution A (saline solution), 8 ml solution C (glucose solution), 10 ml solution D (casein solution).
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.


PREINCUBATION TEST (PIT): 3 test plates per dose or per control
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al. 0.1 ml test solution or vehicle, 0.1 ml bacterial
suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.


TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (vehicle only) and after adding the two highest amounts of substance. For this purpose, in the standard plate test 0.1 ml of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45°C, and the remaining components are added in the following order:
0.1 ml vehicle (without and with test substance)
0.1 ml bacterial suspension (dilution : 10-6 )
0.5 ml S-9 mix
In the PIT, 0.1 ml of the overnight cultures is diluted to 10E-6 in each case. 0.1 ml vehicle (with and without test substance), 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for 20 minutes. Subsequently, 2 ml of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added. After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
In general, a substance to be characterized as positive in the bacterial tests has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect was observed at doses ≥ 2500 ug/plate (SPT) or ≥ 500 ug/plate (PIT).
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
≥ 2500 ug/plate (SPT) or ≥ 500 ug/plate (PIT).

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the TS is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here.
Executive summary:

The study was conducted according to OECD TG 471 and 472 in compliance with GLP and is reliable without restrictions.

The TS was tested for mutagenicity in the Ames test and in the E. coli - reverse mutation assay both in the standard plate test (SPT) and in the preincubation test (PIT) with and without the addition of a metabolizing system obtained from rat liver (S-9 mix) using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. Dose ranges were 20 - 5000 µg/plate (SPT) and 20 - 2000 µg/plate (PIT), respectively.

An increase in the number of his+ or trp+ revertants was not observed both in the SPT and in the PIT either without S-9 mix or after the addition of a metabolizing system.