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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
Study period:
05 January 1994 to 17 January 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Commission Directive 92/69/EEC
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Method

Target gene:
Histidine and tryptophan
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-induced S9
Test concentrations with justification for top dose:
- Preliminary toxicity screen: 0, 312.5, 625, 1250, 2500, 5000 μg/plate.
- Experiment 1: 0, 8, 40, 200, 1000, 5000 μg/plate.
- Experiment 2 (TA100 and WP2uvrA): 0, 156.25, 312.5, 625, 1250, 2500, 5000 μg/plate.
- Experiment 2 (TA1535, TA98 and TA1537): 62.5, 125, 250, 500, 1000, 2000 μg/plate.

Vehicle / solvent:
Sterile distilled water
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG (2 μg/plate for WP2uvrA; 3 μg/plate for TA100; 5 μg/plate for TA1535 without metabolic activation)
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
9AA (80 μg/plate for TA1537 without metabolic activation)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4NQO (0.2 μg/plate for TA98 without metabolic activation)
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2AA (2 μg/plate for TA1535 and 10 μg/plate for WP2uvrA with metabolic activation)
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
BP (5 μg/plate for TA100, TA1537 and TA98 with metabolic activation)
Details on test system and experimental conditions:
TESTER STRAINS
- Strains were obtained from the British Industrial Biological Research Association on 14 August 1987 and were stored at -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).
- Prior to being used, characterisation checks were performed to determine the amino acid requirement, presence of rfa, R factors, uvr mutation and the spontaneous reversion rate.
- In this assay, overnight subcultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited, lot number 35051940 11/97) and incubated at 37 °C for 10 hours.

PREPARATION OF TEST AND CONTROL MATERIALS
- Test item was accurately weighed (with an allowance made for purity) and dissolved in sterile distilled water. Appropriate dilutions were then made.
- The concentration, homogeneity and stability of the test material formulations were not determined by analysis.
- Negative and positive controls were used in parallel with the test material.

MICROSOMAL ENZYME FRACTION
- Lot number Aro S9/02/12/93 prepared on 02 December 1993 was obtained from the British Industrial Biological Research Association on 14 December 1993.
- The microsomal enzyme fraction was prepared from the livers of male Sprague-Dawley rats weighing approximately 200 g. The animals had received an i.p. injection of Aroclor 1254 at 500 mg/kg five days before S9 preparation.
- The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).

S9 MIX AND AGAR
- The S9 mix was prepared at 4 °C.
- S9 mix contained S9 (5.0 mL), 1.65 M KCl/0.4 M MgCl2 (1.0 mL), 0.1 M glucose-6-phosphate (2.5 mL), 0.1 M NADP (2.0 mL), 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL) and sterile distilled water (14.5 mL).
- Top agar was prepared using 0.6 % Difco Bacto agar (lot 23050 12/97) and 0.5 % sodium chloride.
- For the Salmonella strains, 1.0 mM histidine/1.0 mM biotin solution (5 mL) was added to each 100 mL of top agar.
- For the Escherichia coli strain 1.0 mM tryptophan (5 mL) was added to each 100 mL of top agar.
- Base agar plates were prepared using 1.2 % Oxoid Agar Technical No 3 (lot number B224 70671 5/98) with Vogel-Bonner Medium E and 20 mg/mL D-glucose.

PRELIMINARY TOXICITY STUDY
- In order to select the appropriate dose levels for use in the main study, a preliminary test was performed to determine the toxicity of the test material to the tester organisms.
- TA100 or WP2uvrA bacterial suspension (0.1 mL), molten trace histidine or tryptophan supplemented media (2 mL), test solution (0.1 mL) and phosphate buffer (0.5 mL) were overlayed onto sterile plates of Vogel-Bonner minimal agar (30 mL/plate).
- Five doses of the test material and a solvent control were tested in duplicate.
- The plates were scored for revertant colonies and examined for thinning of the background lawn after 48 hours incubation at 37 °C.

MUTATION STUDY - EXPERIMENT 1
- Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
- Plates were incubated at 37 °C for 48 hours and the number of revertant colonies counted.

TEST MATERIAL AND NEGATIVE CONTROLS
- Aliquots (0.1 mL) of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by molten trace histidine or tryptophan supplemented top agar (2.0 mL) at 45 °C. These sets comprised two test tubes for each bacterial tester strain.
Appropriately diluted test material (0.1 mL) or negative control solution was also added to each of the two tubes, followed by S9 liver microsome mix (0.5 mL) or pH 7.4 buffer (0.5 mL).
- The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate).
- The procedure was repeated in triplicate for each bacterial strain and each concentration of test material.

POSITIVE CONTROLS - WITHOUT ACTIVATION
- One of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing molten trace histidine or tryptophan supplemented top agar (2.0 mL) and the appropriate bacterial suspension (0.1 mL).
- Finally, pH 7.4 buffer (0.5 mL) was added to the tube. The contents were then mixed and poured onto agar plates.
- The procedure was then repeated in triplicate for each of the positive controls.

POSITIVE CONTROLS - WITH ACTIVATION
- 2AA or BP solution (0.1 mL) was added to a test tube containing molten trace histidine or tryptophan supplemented top agar (2.0 mL) and the appropriate bacterial suspension (0.1 mL).
- Finally, S9 mix (0.5 mL) was added to the tube. The contents were then mixed and poured onto agar plates.
- The procedure was then repeated in triplicate for each tester strain.

EXPERIMENT 2
- The second experiment was performed in triplicate using fresh bacterial cultures, six concentrations of test material for each strain and control solutions.




Evaluation criteria:
INTERPRETATION OF RESULTS
- For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels.
- If the two experiments give conflicting results then a third experiment may be used to confirm the correct response.
- To be considered negative the number of induced revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 μg/plate. In this investigation the limiting factor was both toxicity and maximum recommended dose.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY STUDY
- Test material was non-toxic in the strains of bacteria used (TA100 and WP2uvrA).
- Mean numbers of revertant colonies for the toxicity assay are shown in the table below.

MUTATION STUDY
- Results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
- Individual plate counts for Experiment 1 are shown in Tables 1 and 2 (attached) together with the mean number of revertant colonies obtained for each tester strain following incubation with test material (with and without metabolic activation).
- Individual plate counts for Experiment 2 are shown in Tables 3 and 4 (attached) together with the mean number of revertant colonies obtained for each tester strain following incubation with test material (with and without metabolic activation).
- Results are expressed graphically in Appendix II (attached).
- Results for positive controls are shown in Tables 1 to 4 (attached).
- Test material caused a reduction in the growth of the bacterial lawn at and above 2000 μg/plate in Salmonella strains TA1535, TA98 and TA1537 both with and without metabolic activation. The test item was therefore investigated up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit of a particular bacterial strain. A precipitate was observed at the maximum dose but this did not interfere with scoring of revertant colonies.
- No significant increases in the numbers of revertant colonies of bacteria were recorded at any dose level for any of the strains of bacteria used (with or without metabolic activation).
- The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Any other information on results incl. tables

Mean numbers of revertant colonies in the preliminary toxicity study

Stain

Dose (μg/plate)

0

312.5

625

1250

2500

5000

TA100

110.0

140.0

123.0

123.5

119.5

102.5

WP2uvrA

36.5

27.0

38.5

31.5

34.5

26.5 (P)

(P) = precipitate

Applicant's summary and conclusion

Conclusions:
No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test.
Executive summary:

In accordance with OECD 471 and EU Method B.14, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA were treated with test material by the Ames plate incorporation method at five and six dose levels. The investigation was conducted in triplicate with and without addition of rat liver homogenate metabolising system at 10 % in standard co-factors. The dose range was determined in a preliminary toxicity assay and was 8 to 5000 μg/plate in the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In this case, various dose ranges of test material were used to allow for differential toxicity to the bacterial strains tested.

The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system. The test item caused a reduction in the growth of the bacterial lawn at and above 2000 μg/plate in Salmonella strains TA1535, TA98 and TA1537 with and without metabolic activation. The test item was therefore tested up to either the maximum recommended dose level of 5000 μg/plate or the toxic limit of a particular bacterial strain. A precipitate was observed at the maximum dose but did not interfere with the scoring of revertant colonies.

No significant increase in the numbers of revertant colonies was recorded with or without metabolic activation for any of the bacterial strains with any dose of test item. The test material was found to be non-mutagenic under the conditions of the test.