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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:
other: read-across from supporting substance (structural analogue or surrogate)
Remarks:
Test substance is one component of the target (UVCB) substance; it is one of the major components, ranging between 10 - 60 % w/w. Other components show slight differences in terms of position / type of functional groups.
Adequacy of study:
key study
Study period:
From January 12 to 27, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Source study has reliability 2, as only 4 strains of Salmonella typhimurium were included, with respect to the 5 strains required by the currently accepted version of the guideline. Details on the read across are available in section 13.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1983
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

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:
liver microsomal activation
Test concentrations with justification for top dose:
33.3, 100.0, 333.3, 1000.0, 2500.0 and 5000.0 µg/plate as a.i., based on results of a pre-experiment on toxicity.
Vehicle / solvent:
- Vehicle: water
The solvent was chosen because of the solubility properties.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
other: 4-nitro-o-phenylene-diamine
Remarks:
without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
THE TEST SYSTEM
Characterisation of the Salmonella typhimurium strains
The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally, due to the "deep rough" (rfa-minus) mutation, they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98 and TA 100 the R-factor plasmid pKM 101 carries the ampicillin resistance marker.
In summary, the mutations of the TA strains used in this study can be described as:
- frame shift mutations
TA 1537: his C 3076; rfa-; uvrB-;
TA 98: his D 3052; rfa-; uvrB-; R-factor
- base-pair substitutions
TA 1535: his G 46; rfa-; uvrB-;
TA 100: his G 46; rfa-; uvrB-;R-factor

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 ul ampicillin was added to the strains TA 98 and TA 100. This nutrient medium contains per litre:
8 g Merck Nutrient Broth
5 g NaCl
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium.
Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar
6.0 g NaCl
10.5 mg L-histidine × HCl × H2O
12.2 mg biotin
Sterilisations were performed at 121 °C in an autoclave.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The bacteria used in these assays do not possess the enzyme systems which, in mammals, are known to convert promutagens into active DNA damaging metabolites. In order to overcome this major drawback an exogenous metabolic system is added in form of mammalian microsome enzyme activation mixture.
S9 (Preparation by C C R)
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain WU, which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for only one week before use.
The protein concentration in the S9 preparation was 37.4 mg/ml.

S9 mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15 % v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures.

DOSE SELECTION
According to the results of this pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered 2 logarithmic decades.
The maximum concentration was 5000.0 µg/plate. The concentration range included two logarithmic decades. In this study 6 adequately spaced concentrations were tested. Two independent experiments were performed.
As the results of the pre-experiment were in accordance with the criteria for evaluation of results, these data are reported as a part of the main experiment I.
According to the dose selection criteria, test substance was tested at the following concentrations:
33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate as active ingredient.

EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls 3 plates were used as a minimum. The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation),
100 µl bacteria suspension,
2000 µl overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and incubated at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45°) was added to each tube.
The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
EVALUATION OF RESULTS
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies (values referred to negative control without metabolic activation and represent historical control range in 1993)
1535 1537 98 100
10 - 29 5 - 28 15 - 57 77 - 189

Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix: 100 µg/plate (exp.I); up to 1000 µg/plate (exp. II)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without S9 mix: at 100 µg/plate (exp. I)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Test substance was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate.
Slight toxic effects occurred in strains TA 1535 and TA 1537 at 100 µg/plate without S9 mix in experiment I. In experiment II, toxic effects occurred in strain TA 1535 up to 1000 µg/plate without S9 mix.
The plates incubated with test substance showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Slight increases in revertant colony numbers were observed in strain TA 1537 at 5000.0 µg/plate without S9 mix and at 2500.0 µg/plate with S9 mix as well as in strain TA 98 at 5000.0 µg/plate with S9 mix in experiment II. These effects are considered being biologically irrelevant since they are due to the relatively low level of the corresponding solvent control (strain TA 1537) and could not be reproduced in the independent experiment (strain TA 1537 and TA 98).
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.
In conclusion, test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Any other information on results incl. tables

Without S9 mix

conc. µg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II
negative control 14 11 8 7 32 18 94 112
solvent control 17 12 12 8 32 16 91 112
33.3 14 5 10 10 28 17 97 102
100.0 8 8 6 10 29 15 106 115
333.3 17 6 11 8 34 14 103 117
1000.0 16 10 9 9 26 18 94 108
2500.0 13 9 13 11 20 16 107 112
5000.0 12 13 15 16 26 13 118 112
positive control
sodium azide 10 µg/plate
741 350 710 337
4-nitro-o-phenylene-diamine
10 µg/plate
38 35 186 376

With S9 mix

conc. µg/plate TA 1535 TA 1537 TA 98 TA 100
I II I II I II I II
negative control 12 10 23 10 45 21 133 141
solvent control 15 14 21 8 41 19 134 136
33.3 15 14 22 9 36 20 120 133
100.0 16 13 22 9 35 24 140 153
333.3 12 11 16 10 39 23 118 149
1000.0 14 13 27 9 35 26 137 162
2500.0 13 11 29 18 38 31 126 149
5000.0 12 11 30 15 46 55 128 158
positive control
2-amino-anthracene (2.5 µg/plate)
62 80 86 12 177 85 483 299

Applicant's summary and conclusion

Conclusions:
Under experimental conditions, test substance did not induce gene mutations by base pair changes or frameshifts in the genome of TA98, TA 100, TA1535, TA 1537 strains.
Executive summary:

Method

The potential of test substance to induce gene mutations was assessed in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100.

The assay was performed in two independent experiments, both with and without liver microsomal activation. Solvent controls, untreated (negative) controls and positive controls were run. Each concentration, including controls, was tested in triplicate. Test substance was dissolved in water and was tested at concentrations of 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate as active ingredient.

Results

Slight toxic effects occurred in strains TA 1535 and TA 1537 at 100 µg/plate without S9 mix in experiment I. In experiment II, toxic effects occurred in strain TA 1535 up to 1000 µg/plate without S9 mix.

The plates incubated with test substance showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

Slight increases in revertant colony numbers were observed in strain TA 1537 at 5000.0 µg/plate without S9 mix and at 2500.0 µg/plate with S9 mix as well as in strain TA 98 at 5000.0 µg/plate with S9 mix in experiment II. These effects were considered as biologically irrelevant, due to the relatively low level of the corresponding solvent control (strain TA 1537) and could not be reproduced in the independent experiment (strain TA 1537 and TA 98).

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Overall, test substance was considered as non-mutagenic in the Salmonella typhimurium reverse mutation assay.