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Toxicological information

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:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(only four strains tested, 2-aminoanthracene was the sole indicator of the efficacy of the S9-mix)
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Name of test material (as cited in study report): C13 - C15 - Alkohol
- Physical state: colorless liquid
- Analytical purity: 99.3% alcohols (0.5% low boiling substances, 0.2% high boiling substances)
- Composition of test material, percentage of components: 29.5% i-C13-alcohol, 33% n-C13-alcohol, 19% i-C15-alcohol, 17.8% n-C15-alcohol
- Lot/batch No.: from continuous production
- Sustance number: 88/577
- Storage: room temperature

Method

Target gene:
his-gene
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of Aroclor 1254 induced rat livers (Sprague-Dawley)
Test concentrations with justification for top dose:
20 - 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in DMSO.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see Details on test system and conditions for details
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

Standard plate test:
The experimental procedure is based on the method of Ames et al ., 1975

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
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.

Preincubation test:
The experimental procedure is based on the method described by Yahagi et al. (1977) and Matsushima et al. (1980).

0.1 ml test solution, 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 Vogel-Bonner 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 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Positive controls:
The following positive control substances are used to check the mutability of the bacteria and the activity of the S-9 mix:

with S-9 mix:
10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535

without S-9 mix:
5 µg N-methyl-N-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535,
10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98,
100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537

NUMBER OF REPLICATIONS: triplicate




Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control);
- dose-response relationship;
- reproducibility of the results;

Results and discussion

Test results
Key result
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Standard plate test (20 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 23 1.0 (20/500/2500/5000) no negative 29 (NPD)
  yes 34 1.1 (20/100) no negative 34.7 (2-AA)
TA 100 no 114 0.9 (100) no negative 14.8 (MNNG)
  yes 103 1.3 (100) no negative 17.2 (2-AA)
TA 1537 no 9 1.3 (20) no negative 47 (AAC)
  yes 12 0.9 (20/100/500) no negative 11.7 (2-AA)
TA 1535 no 15 1.0 (20) no negative 128.4 (MNNG)
  yes 15 1.2 (2500) no negative 10.5 (2-AA)
Preincubation test (20 - 5000 µg/plate)
Strain Metabolic activation system mean his+/trp+revertant colonies (negative control) maximum revertant factor (conc. (µg/plate)) dose dependency Assessment maximum revertant factor (positive control)
TA 98 no 24 1.1 (500) no negative 39.9 (NPD)
  yes 35 1.2 (500) no negative 26.5 (2-AA)
TA 100 no 110 1.1 (500) no negative 10.6 (MNNG)
  yes 125 1.1 (20) no negative 9.5 (2-AA)
TA 1537 no 8 1.2 (100) no negative 43.9 (AAC)
  yes 12 0.8 (20/100/2500) no negative 9.3 (2-AA)
TA 1535 no 19 1.2 (5000) no negative 59 (MNNG)
  yes 18 1.0 (20/5000) no negative 7.1 (2-AA)
2-AA = 2-aminoanthracene
NPD = N-nitro-o-phenylendiamine
MNNG = N-methyl-N-nitro-N-nitrosoguanidine
AAC = 9-aminoacridine chloride monohydrate

Applicant's summary and conclusion