[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:

From April 28th to May 4th, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

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:

July 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite

Molecular formula:

Not applicable - Multiconstituent substance

IUPAC Name:

Reaction mass of dodecane-1-thiol and tridodecyl trithiophosphite

Test material form:

liquid

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

The concentration used in the first experiment (plate incorporation method) and in the second experiment (pre-incubation method) were the same , i.e. : 5.0, 2.50, 1.25, 0.625 and 0.313 µl/plate. This concentrations were chosen based on the results of the preliminary toxicity test performed at concentration 5.00, 1.58, 0.50, 0.158, 0.05 µl/plate. In the preliminary test nor precipitation of the test item neither relevant toxicity or relevant increase in revertant colonies was observed.

Vehicle / solvent:

- Solvent(s) used: acetone to dissolve the test item, DMSO or water for injection to dissolve positive controls.
- Justification for choice of solvent/vehicle: solubility of the test item was evaluated in a preliminary trial using DMSO, ethanol and acetone. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be fully miscible in acetone at 50 %v/v. This result permitted a maximum concentration of 5 µL/plate to be used in the toxicity test.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9

Details on test system and experimental conditions:

BACTERIA STRAIN
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work. Bacteria were taken from vials of frozen cultures, which had been checked for the presence of the appropriate genetic markers, as follows:
- Histidine requirement: No Growth on Minimal plates + Biotin and Growth on Minimal plates + Biotin + Histidine.
- Tryptophan requirement: No Growth on Minimal agar plates and Growth on Minimal plates + Tryptophan.
- uvrA, uvrB: Sensitivity to UV irradiation.
- rfa: Sensitivity to Crystal Violet.
- pKM101: Resistance to Ampicillin.

MEDIA
- Nutrient Broth: Oxoid Nutrient Broth No. 2 was prepared at a concentration of 2.5% in distilled water and autoclaved prior to use. This was used for the preparation of liquid cultures of the tester strains.
- Nutrient Agar: Oxoid Nutrient Broth No. 2 (25 g) and Difco Bacto-agar (15 g) were added to distilled water(1 litre) and autoclaved. The solutions were then poured into 9 cm plastic Petri dishes and allowed to solidify and dry before use. These plates were used for the non-selective growth of the tester strains.
- Minimal Agar: Minimal medium agar was prepared as 1.5% Difco Bacto-agar in Vogel-Bonner Medium E, with 2% Glucose, autoclaved and poured into 9 cm plastic Petri dishes.
- Top Agar: "Top Agar" (overlay agar) was prepared as 0.6% Difco Bacto-agar + 0.5% NaCl in distilled water and autoclaved. Prior to use, 10 ml of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5 mM tryptophan) was added to the top agar (100 ml).

S9 TISSUE
One batch of S9 tissue fraction was used in this study and had the following characteristics:
- Species: Rat
- Strain Sprague: Dawley
- Tissue: Liver
- Inducing Agents: Phenobarbital – 5,6-Benzoflavone
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 ml): S9 tissue fraction 1.0 ml+ NADP (100 mM) 0.4 ml+G-6-P (100 mM) 0.5 ml+ KCl (330 mM) 1.0 ml+ MgCl2 (100 mM) 0.8 ml+ Phosphate buffer (pH 7.4, 200 mM) 5.0 ml+ Distilled Water 1.3 ml

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the Main Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

MAIN ASSAYS
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures.
- FIRST MAIN ASSAY -PLATE INCORPORATION METHOD: the components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The overlay mixture was composed as follows: overlay agar (held at 45°C) 2.0 ml +solvent/ test or control item solution 0.03 ml/ 0.1 ml+ S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml+ Bacterial suspension 0.1 ml.
- SECOND MAIN ASSAY- PRE-INCUBATION METHOD: the components were added in turn to an empty test-tube: bacterial suspension 0.1 ml+ solvent /vehicle or test item solution 0.01 ml or control item solution 0.05 ml+ S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 ml. The incubate was vortexed and placed at 37°C for 30 minutes. Two ml of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates from the preliminary toxicity test were held at 4°C for approximately 24 hours before scoring, while plates from Main assay were immediately scored by counting the number of revertant colonies on each plates.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
- Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
- The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
- No more than 5% of the plates should be lost through contamination or other unforeseen event.

Evaluation criteria:

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Statistics:

The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a +bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation coeffi cient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Key result

Species / strain:

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

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:

RESULTS OF PLATE INCORPORATION AND PRE-INCUBATION ASSAY:
On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5.00, 2.50, 1.25, 0.625 and 0.313 μl/plate.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.
No precipitation of the test item was observed at the end of the incubation period at any concentration, in any experiment.
Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

TOXICITY PRELIMINARY TEST
The test item was assayed in the toxicity test at a maximum dose level of 5.00 μl/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 μ/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration.Neither relevant toxicity, nor relevant increase in revertant colonies was observed, with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

ACCEPTANCE CRITERIA:
All acceptance criteria were fulfilled and the study was accepted as valid:
- results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data;
- the estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain;
- no plates were lost through contamination or cracking.

EVALUATION OF RESULT:
The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism. It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Applicant's summary and conclusion

Conclusions:

The test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.The test item was used as a solution in acetone.

 

TOXICITY TEST

The test item was assayed in the toxicity test at a maximum concentration of 5.00 µl/plate and at four lower concentrations spaced at approximately half-log intervals: 1.58, 0.500, 0.158 and 0.0500 µl/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither relevant toxicity,nor relevant increase in revertant colonies was observed, with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

 

MAIN ASSAY

On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5.00, 2.50, 1.25, 0.625 and 0.313 µl/plate. No toxicity was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.

No precipitation of the test item was observed at the end of the incubation period at any concentration in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

 

CONCLUSION

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.