[No public or meaningful name is available]

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:

12 February to 02 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 471 Guideline without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.15 (Gene Mutation - Saccharomyces cerevisae)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other:

Version / remarks:

24 November 2000

Deviations:

no

Remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of 28 November 2017

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Storage: room temperature
Form: liquid
Colour: colorless to yellowish

Specific details on test material used for the study:

Identification: Socogel part I
Purity: Confirmed as a mixture and treated as 100%
Physical state/Appearance: Pale straw coloured liquid

Method

Target gene:

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.
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.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male Wistar rats treated with Phenobarbital/ß-naphthoflavone (80 mg/kg bw/day by oral route).

Test concentrations with justification for top dose:

Experiment I (plate incorporation test):
In the pre-experiment, eight concentrations of the test item between 3 and 5000 μg/plate were tested as followed:
- All strains: 1.5, 5; 15; 50; 150; 500; 1500; 5000 μg/plate.
Experiment II : (pre-incubation test):As a clear negative response was obtained in Experiment 1, a variation to the test procedure was used for the second test. Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
The dose range used for Experiment 2 was determined following the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.

Vehicle / solvent:

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.
In solubility checks performed in house, the test item was only partially miscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxyde

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (2AA)

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulphoxyde

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

TEST SYSTEM:
The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH and British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
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.

METHOD OF APPLICATION:
Experiment I in agar (plate incorporation)
Experiment II : pre-incubation

DURATION
- Preincubation period: at 37°C +/-3°C for 20 minutes
- Exposure duration: plates were incubated upside down for at least 48 hours at 37°C +/-3°C

SELECTION AGENT (mutation assays): Plates with selective agar (without histidine/tryptophan) were used.

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHERS:
- The colonies were counted using a validated computer system.

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:
A dose-related increase in mutant frequency over the dose range tested.
A reproducible increase at one or more concentrations.
Biological relevance against in-house historical control ranges.
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
If exposure to a test item produces a reproducible increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it will be considered to exhibit mutagenic activity in this test system (Cariello and Piegorsch, 1996)).
If exposure to a test item does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system.

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. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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

Test results 3

Key result

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

Test results 4

Key result

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

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

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-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not reported
- Effects of osmolality: Not reported
- Evaporation from medium: Not reported
- Water solubility: Not reported
- Precipitation: In the first experiment, employing the plate incorporation method, there was no precipitate noted at any test item concentration in either the absence or presence of S9-mix. However, in Experiment 2, which utilized the pre-incubation modification, a test item precipitate was noted in both the absence and presence of S9-mix initially under a low power microscope at 500 µg/plate and by eye from 1500 µg/plate. The precipitate observation did not affect the scoring of revertant colonies.
- Other confounding effects: Not reported

Combined historical negative and solvent control ranges for 2016 and 2017

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first experiment, the maximum dose level of the test item was selected as 5000 µg/plate (the maximum recommended concentration). There was no visible reduction in the growth of the bacterial background lawns noted to any of the tester strains in either the absence and presence of S9-mix at any test item dose level. Consequently, in the second experiment, the maximum recommended dose level (5000 µg/plate) was employed as the maximum concentration. Once again, no visible reduction in the growth of the bacterial background lawns was noted to any of the tester strains in either the absence and presence of S9-mix at any test item dose level.

MUTAGENICITY RESULTS
See 'background material'

Applicant's summary and conclusion

Conclusions:

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 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).  

It was concluded that Socogel part I was considered to be non-mutagenic up to and including 5000 µg/ plate under the conditions of this test.

Executive summary:

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.

Histidine-dependent auxotrophic mutants of Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and a tryptophan-dependent mutant of Escherichia coli strain WP2uvrA were treated with 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 g/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 per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology.  

The vehicle (dimethyl sulphoxide) 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.

In the first experiment (plate incorporation method), the maximum dose level of the test item was selected as 5000 µg/plate (the maximum recommended concentration).  There was no visible reduction in the growth of the bacterial background lawns noted to any of the tester strains in either the absence and presence of S9-mix at any test item dose level.  

In the second experiment (pre incubation method), the same maximum recommended dose level (5000 µg/plate) was employed as the maximum concentration.  Once again, no visible reduction in the growth of the bacterial background lawns was noted to any of the tester strains in either the absence and presence of S9-mix at any test item dose level.  

In the first experiment, employing the plate incorporation method, there was no precipitate noted at any test item concentration in either the absence or presence of S9-mix.  However, in Experiment 2, which utilized the pre-incubation modification, a test item precipitate was noted in both the absence and presence of S9-mix initially under a low power microscope at 500 µg/plate and by eye from 1500 µg/plate.  The precipitate observation did not affect 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 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).  

It was concluded that Socogel part I was considered to be non-mutagenic up to and including 5000 µg/ plate under the conditions of this test.