Butanedioic acid, sulfo-, monoesters with lanolin alcs., disodium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

between 25 February to 26 March 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Data have been obtained from a study on a substance with structural similarity. GLP Compliance statement (UK GLP standards); No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. GLP standards published as OECD (ENV/MC/CHEM(98)17); Directives 2004/9/EC and 2004/10/EC

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: USA, EPA (TSCA) OPPTS harmonized guidelines

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Lanolin Alcohols

IUPAC Name:

Lanolin Alcohols

Test material form:

other: solid

Method

Target gene:

Using various strains Salmonella, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is either added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional traits have been introduced. These include a deletion through the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall.
A mutant strain of Escherichia coli (WP2uvrA-) can be reverse mutated by base substitution to tryptophan independence was used to complement the Salmonella strains. This strain also has a deletion in an excision repair gene (uvrA-).

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: When exposed to a mutagenic agent, mutants of Salmonella may undergo a reverse to histidine independent forms which they are detected by their ability to grow on a histidine deficient medium. Obtained in culture discs or frozen vials.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: Escherichia Coli requires tryptophan for growing. Obtained on a nutrient agar plate.

Metabolic activation:

with

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Formulated concentrations were adjusted to allow for the stated water/impurity content (10%) of the test material.
No analysis was conducted to determine the homogeneity, concentration or stability of the test material formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Vehicle / solvent:

Acetone.

Controls

Controls 1

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Controls 2

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

2AA and BP were used in series of plates with S9-mix (non-mutagenic in the absence of metabolising enzymes)

Details on test system and experimental conditions:

STORAGE OF STRAINS:
All of the stains were stored approx. at - 196 °C in a Statebourne liquid Nitrogen freezer, model SXR 34.
Characterization checks were carried out to confirm the amino-acid requirement presence of rfa, R factors, uvrB or uvrA mutation and the spontaneous reversion rate.

PREINCUBATION PERIOD OF STRAINS:
overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 757012 03/14) 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.

PREPARATION OF TEST MATERIAL
Test material (fully soluble in acetone) was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer and sonication for 1 minute at 40°C on the day of each experiment.
Prior to use, the solvent was dried to remove water using molecular sieves ie 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns. No analysis was carried out to determine the homogeneity, concentration or stability of the test material formulation.

MICROSOMIAL ENZYME FRACTION
S9 was prepared in-house on 06 December 2009 from the livers of male rats weighing approximately 2509. These had each orally received three consecutive daily doses of phenobarbitone/ ß-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on day 4. Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9 was stored at approximately -196 °C.

S9-MIX
The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCI/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
Sterile distilled water 14.5 mL
A 0.5 ml aliquot of S9-mix and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

AGAR
Top agar was prepared using 0.6% Bacto agar (lot number 9070630 01/14) and 0.5% sodium chloride with 5 ml of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 ml of top agar.

TEST PROCEDURE:
1) PRELIMINARY TOXICITY TEST
In order to select appropriate dose levels for use in the main test,
The concentrations tested: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
The test was performed by mixing 0.1 ml of bacterial culture (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of test material formulation and 0.5 ml of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 ml/plate).
Ten concentrations of the test material formulation and a vehicle control (acetone) were tested.
In addition, 0.1 ml of the maximum concentration of the test material and 2 ml of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Nutrient agar plate in order to assess the sterility of the test material. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn. Manual counts were performed at 5000 μg/plate because of test material precipitation.
2) EXPERIMENT 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer.
The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate).
This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at 5000 pg/plate because of test material precipitation.
3) EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 μg /plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 ml of S9-mix or phosphate buffer and 0.05 ml of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 ml of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method. All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter. Manual counts were performed at and above 1500 μg/plate because of test material precipitation.

Acceptance criteria: all tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls. The appropriate characteristics for each tester strain have been confirmed.
All tester strain cultures should be in the range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix. There should be a minimum of four non-toxic test material dose levels. There should be no evidence of excessive contamination.

Evaluation criteria:

Evaluation criteria:
biological relevance of the results;

Statistics:

Statistical significance will not be the only determining factor for a positive response.

Results and discussion

Test results

Test results 1

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

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

1) PRELIMINARY TOXICITY TEST
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).
2) MUTATION TEST
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was tested up to the maximum recommended dose level of 5000 μg/plate. A greasy, particulate precipitate was noted at and above 1500 and 500 μg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9mix and the sensitivity of the bacterial strains.

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Genetic toxicity in vitro was performed by methods conforms to the guidelines:

- Bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF;

- OECO Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test";

- Method 813/14 of Commission Regulation; - (EC) Number 440/2008 of 30 May 2008;

- USA, EPA (TSCA) DPPTS harmonised guidelines.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2uvrA- were treated with the test material using both the Ames plate incorporation and pre-incubation methods at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated (Experiment 2) on a separate day (pre-incubation method) using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations. The vehicle (acetone) 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 test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A greasy, particulate precipitate was noted at and above 1500 and 500 µg/plate in Experiments 1 and 2, respectively. This observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. As conclusion the test material was considered to be non-mutagenic under the conditions of this test.