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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 16th to 30th, 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone.
Species / strain:
E. coli WP2 uvr A
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) was selected considering the results of preliminary toxicity test as follows:
TA1535 (without metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate
TA98 (with metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate
TA 1537 and TA98 (without metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate
TA 1535 and TA1537 (with metabolic activation): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
WP2 uvrA (with and without metabolic activation): 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA 100 (with and without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

The concentration used in the second experiment (pre-incubation method) was modified taken in account the results of first assay as follows:
TA1535 (without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA 100 (with metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA1535 (with metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate
TA 1537 and TA98 (without metabolic activation): 4.88, 9.77, 19.5, 39.1, 78.1 and 156 µg/plate
TA 1537 and TA98 (with metabolic activation): 39.1, 78.1, 156, 313, 625 µg/plate
WP2 uvrA (with and without metabolic activation): 1.22, 2.44, 4.88, 9.77, 19.5 and 39.1 µg/plate
TA 100 (without metabolic activation): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate.
Vehicle:
- Solvents used: Dimethylsulfoxide (DMSO) for 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. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. Using DMSO, an opaque dispersion with precipitation was obtained at 12.5 mg/ml following magnetic stirring, while a clear solution without any visible precipitate was observed at 6.25 mg/ml. This result permitted a maximum concentration of 1250 µg/plate to be used in the toxicity test.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Remarks:
Without S9
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9
Details on test system and 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 0.1 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 0.05 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 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 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Toxicity as indicated by microcolony formation, clearance of the back-ground lawn and/or reduction in revertant colonies, were observed in both experiments at higher dose levels for many strains with and without metabolic activation.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF PLATE INCORPORATION AND PRE-INCUBATION ASSAY:
Two Main Assays were performed. The maximum concentration of the test item to be used in the main experiments was selected as the concentration which elicits a moderate toxicity. The number of lower dose levels included in each treatment series was selected in order to have a sufficient number of analysable concentrations. 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:
TA1535 (without metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate
TA98 (with metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate
TA 1537 and TA98 (without metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate
TA 1535 and TA1537 (with metabolic activation): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate
WP2 uvrA (with and without metabolic activation): 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate
TA 100 (with and without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
Dose related toxic effects, as indicated by microcolony formation, clearance of the back-ground lawn and/or reduction in revertant colonies, were observed at higher dose levels with all tester strains, with the exception of TA1535, TA1537 and TA98 in the presence of S9 metabolism.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. Due to the DMSO toxicity, a lowest dose volume was used in the preparation of each plate, therefore the maximum practicable concentration was 625 µg/plate. In addition the dose range used was modified to take into account the toxicity observed in the first assay and the following dose levels were used:
TA1535 (without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA 100 (with metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate
TA1535 (with metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate
TA 1537 and TA98 (without metabolic activation): 4.88, 9.77, 19.5, 39.1, 78.1 and 156 µg/plate
TA 1537 and TA98 (with metabolic activation): 39.1, 78.1, 156, 313, 625 µg/plate
WP2 uvrA (with and without metabolic activation): 1.22, 2.44, 4.88, 9.77, 19.5 and 39.1 µg/plate
TA 100 (without metabolic activation): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate.
Toxicity, as indicated by thinning of the background lawn, was observed at the highest or two highest dose levels with all tester strains in the absence of S9 metabolism. In its presence, a slight toxic effect was observed with WP2 uvrA only. No increase in revertant numbers was noted with any tester strain/activation condition combination.
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.
No precipitation of the test item was observed at the end of the incubation period in the absence or presence of S9 metabolic activation, in any experiment.

TOXICITY PRELIMINARY TEST
The test item Tetraphosphorus trisulphide was assayed in the toxicity test at a maximum dose level of 1250 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 395, 125, 39.5 and 12.5 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Dose related toxic effects, as indicated by microcolony formation, thinning of the background lawn and/or reduction in revertant numbers, were observed both in the absence and presence of S9 metabolism with almost all tester strains. A more pronounced toxic effect was noted with WP2 uvrA showing thinning of the background lawn up to 39.5 µg/plate. No relevant increase in revertant numbers was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

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:
Tetraphosphorus trisulphide does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.
Executive summary:

The test item Tetraphosphorus trisulphide 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 dispersion in dimethylsulfoxide (DMSO).

Toxicity test

The test item Tetraphosphorus trisulphide was assayed in the toxicity test at a maximum concentration of 1250 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 395, 125, 39.5 and 12.5 µg/plate. Dose related toxic effects, ranging from slight to complete toxicity, were observed both in the absence and presence of S9 metabolic activation with almost all tester strains. A more pronounced toxic effect was noted with WP2 uvrA tester strain. No precipitation of the test item was observed at the end of the incubation period at any concentration tested.

Main Assays

Based on 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:

TA1535 (without metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate

TA98 (with metabolic activation): 78.1, 156, 313, 625, 1250 µg/plate

TA 1537 and TA98 (without metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate

TA 1535 and TA1537 (with metabolic activation): 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

WP2 uvrA (with and without metabolic activation): 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate

TA 100 (with and without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

Toxicity was observed at higher dose levels, both in the absence and presence of S9 metabolic activation with all tester strains, with the exception of TA1535, TA1537 and TA98 in the presence of S9 metabolism.

 

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. Due to the DMSO toxicity, a lowest dose volume was used in the preparation of each plate, therefore the maximum practicable concentration was 625 µg/plate. In addition the dose range used was modified to take into account the toxicity observed in the first assay and the following dose levels were used:

TA1535 (without metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

TA 100 (with metabolic activation): 9.77, 19.5, 39.1, 78.1, 156 and 313 µg/plate

TA1535 (with metabolic activation): 19.5, 39.1, 78.1, 156, 313, 625 µg/plate

TA 1537 and TA98 (without metabolic activation): 4.88, 9.77, 19.5, 39.1, 78.1 and 156 µg/plate

TA 1537 and TA98 (with metabolic activation): 39.1, 78.1, 156, 313, 625 µg/plate

WP2 uvrA (with and without metabolic activation): 1.22, 2.44, 4.88, 9.77, 19.5 and 39.1 µg/plate

TA 100 (without metabolic activation): 2.44, 4.88, 9.77, 19.5, 39.1 and 78.1 µg/plate.

In the absence of S9 metabolism, toxic effects were noted at the highest or two highest concentrations tested with all tester strains. Following treatment in the presence of S9 metabolism, no toxicity was observed with any tester strain, with the exception of WP2 uvrA. No increase in revertant numbers was noted with any tester strain/activation condition combination.

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 Tetraphosphorus trisulphide does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions