Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 APR 2002 - 5 JUL 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD TG 471.

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:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
8 June 2000
Deviations:
yes
Remarks:
Due to a technical error, slightly higher concentrations were evaluated in the current experiments (the quality of the study, however, was not impaired).
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
yes
Remarks:
Due to a technical error, slightly higher concentrations were evaluated in the current experiments (the quality of the study, however, was not impaired).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
HIS operon (S. thyphimurium)
TRY operon (E. coli)
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
- source of S9 : Male Wistar, HSdCpb:Wu rats from Harlan Winkelmann, Borchen, Germany, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) diluted in Miglyol 812 oil. The animals received drinking water and a standard diet ad libitum. About 16 hours before sacrifice, the feed was removed. Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice-cooled, sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer containing 3 ml of 0.15 M KCl per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 minutes at about +4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen in liquid nitrogen and stored at -196°C.
- method of preparation of S9 mix:

Quantity per ml S 9 mix
Components 1st Series 2nd Series

Liver homogenate (S9) 0.10 mL 0.30 mL
MgCQ/KCl aqueous solution (0.4 M/l .64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate, disodium salt 5 µmol 5 µmol
NADP, disodium salt 4 µmol 4 µmol
Sodium phosphate buffer (0.2 M, pH 7.4) 0.50 mL 0.50 mL
Distilled water 0.38 mL 0.18 mL

- concentration or volume of S9 mix and S9 in the final culture medium : 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene are used as an acceptance criterion for each S9-batch. In this study 2-aminoanthracene, and benzo[a]pyrene for TA 102, are used as the concurrent positive controls for the different strains in the presence of S9 mix.
Test concentrations with justification for top dose:
1st series: 5.53, 17.5, 55.3, 175, 553, 1750 and 5530 µg per plate
2nd series: 5.53, 17.5, 55.3, 175, 553, 1750 and 5530 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: distilled water or dimethyl sulfoxide (DMSO) are the preferred solvents

- Justification for percentage of solvent in the final culture medium: Since organic solvents may have diverse effects on e.g. gene regulation usually the maximum amount of solvent is limited to 10 µL per plate for DMSO. Only the highest test material concentration may be plated with 31.6 µL organic solvent.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
cumene hydroperoxide
other: Daunomycin, 2-Aminoanthracene
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
2 - 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored using an Artek MiniCount colony counter or manually. The presence of a background lawn of non-revertant cells was checked for each plate. Tables of individual and mean values are generated by use of a validated, automated data processing program (COLONY V2.31, York Electronic Research, York, UK).

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

-----------------------------------------------------------------------------------------
Mean Number of Colonies Maximal Mean Number of Colonies over the
(Solvent Control) Actual Solvent Control (Test Material)
-----------------------------------------------------------------------------------------

<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200

Assessment No increase Clear increase

-----------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak increases".
Interpretations:
A test material is defined as non-mutagenic in this assay if "no" or "weak increases" occur in the first and second series of the main experiment.
("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:

- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;

- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed.
If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.
Evaluation criteria:
see datails on test system

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(≥ 1750 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations of ≥ 553 µg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative control mutant frequencies were all in the regular range. The strain specific positive control compounds, namely daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine, and cumene hydroperoxide in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene and benzo[a]pyrene, which require metabolic activation, were strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was functioning.

Ames test:
- Signs of toxicity : Toxicity to the bacteria was observed at concentrations ≥1750 µg/plate.
- Mean number of revertant colonies per plate and standard deviation : please refer to file attached under 'Attached background material'

Applicant's summary and conclusion

Conclusions:
With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.
Executive summary:

Study Design

The GLP compliant study was performed in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997). The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1stand 2ndseries, respectively.

Results

The test item  was dissolved in dimethyl sulfoxide (DMSO) and tested at concentrations ranging from 5.53 to 5530 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations ≥ 553 µg/plate. Toxicity to the bacteria was observed at concentrations ≥ 1750 µg/plate.
Daunomycin, N-ethyl-N`-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain.
Thus, the test item was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.