Sulfonic acids, shale-oil, sodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

initiated: 2016-08-22, experimental: 2016-10-20 to 2016-11-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

recommended study/method

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

MHRA, date of issue: 28/10/2016

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic acids, shale-oil, sodium salts

EC Number:

297-668-0

EC Name:

Sulfonic acids, shale-oil, sodium salts

Cas Number:

93686-18-1

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Sulfonic acids, shale-oil, sodium salts

Test material form:

liquid - solid: mixture of

Remarks:

aqueous solution

Details on test material:

Tiroler Steinöl sulfoniert D90 (TS-D90)

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

Method

Target gene:

Histidine is the target gene for S. typhimurium strains.
Tryptophan is the target gene for E. coli strains.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: see materials and methods section

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: see materials and methods section

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (with valid Certificate of S9 Efficacy)

Test concentrations with justification for top dose:

Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

sterile distilled water

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

see tabels 2 to 5

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

see tabels 2 to 5

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

see tabels 2 to 5

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoacridine

Remarks:

see tabels 2 to 5

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

see tabels 2 to 5

Details on test system and experimental conditions:

The purpose of the study was to evaluate Tiroler Steinöl sulfoniert D90 for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

BACTERIA
The five strains of bacteria used, and their mutations, are as follows:
Strains Genotype Type of mutations indicated
Salmonella typhimurium:
TA1537 his C 3076; rfa-; uvrB-: frame shift
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitution
TA100 his G 46; rfa-; uvrB-;R-factor
Escherichia coli:
WP2uvrA trp-; uvrA-: base-pair substitution

All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker. In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1758279 10/20 and 1865318 05/21) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

TEST ITEM PREPARATION AND ANALYSIS
The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle. The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water content (70%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

TEST FOR MUTAGENICITY: EXPERIMENT 1 - PLATE INCORPORATION 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:
0.1 mL of the appropriate concentration of test item, solvent vehicle or 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 then 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 was the same as described previously (see 3.3.2.2) 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 ± 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).

TEST FOR MUTAGENICITY: EXPERIMENT 2 - PRE-INCUBATION METHOD
As Experiment 1 was deemed negative, Experiment 2 was performed using the preincubation method in the presence and absence of metabolic activation.

Dose selection:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 5 to 5000 μg/plate. Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology.

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

With Metabolic Activation:
The procedure was the same as described previously (see 3.3.3.2) 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 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring:
All of the plates were incubated at 37 ± 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). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium.

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:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Major Computerized Systems:
Sorcerer Imaging System with Ames Study Manager and Delta Building Monitoring System.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report. A viability count for strain TA100 was just below the minimum level. This count was still considered acceptable as the other vehicle and untreated control counts were within expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test. The individual plate counts, the mean number of revertant colonies and the standard
deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1.

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. The test item induced a visible reduction in the growth of the bacterial background lawns in the second mutation test after implementation of the pre-incubation method. Weakened bacterial background lawns were noted in the absence of S9-mix from 500 μg/plate (TA100), 1500 μg/plate (TA1535 and TA1537) and at 5000 μg/plate (TA98). In the presence S9-mix weakened bacterial background lawns were noted to two of the Salmonella strains (TA1535 and TA1537) at 5000 μg/plate. No toxic response was noted for Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix and Salmonella strains TA100 and TA98 dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item induced bronze colouration was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 5000 μg/plate in the second mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies. 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.

Applicant's summary and conclusion

Conclusions:

Tiroler Steinöl sulfoniert D90 was considered to be non-mutagenic under the conditions of this test.

Executive summary:

System / Method

This study was performed to investigate the potential of Sulfonic acids, shale oil, sodium salts to induce gene mutations in the plate incorporation test (Experiment 1) and the pre-incubation test (Experiment 2) using the Salmonella Typhimurium strains TA 1535, TA 100, TA 1537 and TA 98, and the Escherichia coli strain WP2 uvrA. The study was conducted according to OECD Guideline 471 and to GLP standard.

The assay was performed in two independent experiments both with and without the addition of a rat liver homogenate metabolizing system. Each concentration, including the controls, was tested in triplicate. The test item was tested at the folowing concentrations:

Experiment 1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 (pre-incubation): 5, 15, 50, 150, 500, 1500 and 5000  µg/plate.

Result

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. The test item induced a visible reduction in the growth of the bacterial background lawns in the second mutation test after implementation of the pre-incubation method. Weakened bacterial background lawns were noted in the absence of S9-mix from 500 µg/plate (TA100), 1500 µg/plate (TA1535 and TA1537) and at 5000 µg/plate (TA98). In the presence S9-mix weakened bacterial background lawns were noted to two of the Salmonella strains (TA1535 and TA1537) at 5000 µg/plate. No toxic response was noted for Escherichia coli strain WP2uvrA in either the absence or presence of S9-mix and Salmonella strains TA100 and TA98 dosed in the presence of S9-mix. The sensitivity of the bacterial tester strains to the

toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item induced bronze colouration was noted at and above 1500 µg/plate, this observation did not prevent the scoring of revertant colonies. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. 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 (pate 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). A small, statistically significant increase in TA1535 revertant colony frequency was observed in the presence of S9-mix at 5000 µg/plate in the second mutation test. This increase was considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore the response would be due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

Conclusion

Sulfonic acids, shale oil, sodium salts, was considered to be non-mutagenic under the conditions of this test.