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Genetic toxicity: in vitro

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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
Study period:
14 May 2015 - 04 June 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Slags, sponge iron production by coal reduction in retort
EC Number:
940-884-3
Molecular formula:
Not applicable for inorganic UVCB
IUPAC Name:
Slags, sponge iron production by coal reduction in retort
Test material form:
solid

Method

Target gene:
Not required
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Non-mammalian study
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Non-mammalian study
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1 - Plate Incorparation Method - 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 - Preincubation Method - 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle/Solvents used : sterile distilled water.
- The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide, polyethylene glycol 400 and acetonitrile at 50 mg/mL, acetone at 200 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in sterile distilled water at a maximum concentration of 25 mg/mL, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 μg/plate for TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 μg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Remarks:
1, 2, 10 µg/plate for TA100, TA1535&TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 μg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

EXPERIMENT 1 - PLATE INCORPARATION METHOD
Dose selection
- The test item was tested using the following method.
- The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
- Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
- 0.2 mL of the appropriate concentration of test item or vehicle or 0.1 mL of the appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer.
- These were mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test.
- Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
- The procedure followed was the same as described above except that following the addition of the test item formulation and bacterial culture; 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
- All of the plates were incubated at 37 °C+/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
- Manual counts were performed at 5000µg/plate due to excessive test item precipitation.
- Addition manual counts were required due to revertant colonies spreading slightly, distorting the plate count.

As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation

EXPERIMENT 2 - PRE-INCUBATION METHOD
Dose selection
- The dose range used for the main test was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.
- Six test item concentration were selected to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
- 0.1mL of appropriate bacterial strain culture, 0.5mL of phosphate buffer and 0.2mL of the test item formulation or solvent or 0.1mL of appropriate positive control were incubated at 37°C ± 3°C for 20 minutes (with shaking) prior to addition of 2mL of molten amino-acid supplemented media and subsqquent plating onto Vogel-Bonner plates.
- Negative (untreated) controls were also performed on the same day as the muttaion test emplying the plate incorparation method.
- All testing was performed in triplicate.


With Metabolic Activation
- The procedure was the same as described above except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C± 3 °C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media.
- All testing for this experiment was performed in triplicate.

Incubation and Scoring
- All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system.
- The plates were viewed microscopically for evidence of thinning (toxicity).
- Manual counts were performed at 5000µg/plate because of excessive test item preciptiation.
- Additional manual counts were rrequired, predominantly due to interference in the base agar; minor precipitation of salts/dust particles/marks on the base of the plates slightly distorting the counts.



NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
All strains 0.9 to 9 * 10>9



ACCEPTANCE CRITERIA:

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60
Evaluation criteria:
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain
(especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this
type will be reported as equivocal.
Statistics:
MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Results and discussion

Test resultsopen allclose all
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

See attached Tables 2 - 5 for results

- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.

- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method)

- Consequently the same maximum dose level was used in the second mutation test.

- Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method).

- A test item precipitate (black specks in appearance) was observed at 5000 μg/plate in Experiment 1 (plate incorporation method) and from 1500 μg/plate in the second mutation test (pre-incubation method). This observation did not prevent the scoring of revertant colonies.

- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

- Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

- Small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 1500 μg/plate (TA100 and TA1535) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls

Applicant's summary and conclusion

Conclusions:
The genetic toxicity of the test item was assessed in accordance with OECD Guideline 471. The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose.

Results

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test item precipitate (black specks in appearance) was observed at 5000 μg/plate in Experiment 1 (plate incorporation method) and from 1500 μg/plate in the second mutation test (pre-incubation method). This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the second mutation test at 1500 μg/plate (TA100 and TA1535) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

Conclusion

Petrit T was considered to be non-mutagenic under the conditions of this test.

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