4-anilinobenzenediazonium hydrogen sulphate

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 September 15th to October 6th, 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:

4-anilinobenzenediazonium hydrogen sulphate

EC Number:

224-756-8

EC Name:

4-anilinobenzenediazonium hydrogen sulphate

Cas Number:

4477-28-5

Molecular formula:

C12H11N3O4S

IUPAC Name:

4-anilinobenzene-1-diazonium hydrogen sulfate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

All manipulations of the test item and treatment steps were performed under safe-light conditions(yellow light) because the test item is photochemically instable.

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 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction prepared from livers of Sprague-Dawley rats pretreated with Phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

On the basis of toxicity test results, in the Main Assay, using the plate incorporation method, the test item was assayed at the following dose levels:
TA1535 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247, 0.123 μg/plate
TA1535 (with S9): 15.8, 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
WP2 uvrA (without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
WP2 uvrA (with S9): 15.8, 7.90, 3.95, 1.98, 0.988 μg/plate
TA1537 (with and without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate
TA100 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate
TA100 (with S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
TA98 (with and without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:solubility of the test item was evaluated in a preliminary trial using water for injection, DMSO and ethanol. These solvents were selected since they are compatible with the survival of the bacteria and the S9 metabolic activity. A clear solution without any visible precipitation was obtained in DMSO at 50.0 mg/ml following few minutes of vortexing. This result permitted a maximum concentration of 5000 µg/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-aminoantracene

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 the laboratory. 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 ASSAY
A single Main Assay was 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.
The Main Assay was performed using a 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, Test or control item solution 0.1ml, S9 mix or phosphate buffer (pH 7.4, 0.1M) 0.5ml, Bacterial suspension 0.1ml.

INCUBATION AND SCORING
The prepared plates were inverted and incubated for approximately 72 hours at 37 °C. After this period of incubation, plates were held immediately scored by counting the number of revertant colonies on each plate.

METHOD OF APPLICATION: single main assay with plate-incorporation method.

ACCEPTANCE CRITERIA:
The assay was considered valid if the following criteria were met:
1.Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
3. 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 co-efficient (r),the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

other: Salmonella typhimurium (TA1537, TA98 and TA100) and Escherichia coli (WP2 uvrA)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity, as indicated by thinning of the background lawn, was observed at the highest or two highest dose levels both in the absence and presence of S9 metabolism with all tester strains, with the exception of WP2 uvrA and TA98.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxicity, as indicated by thinning of the background lawn, was observed at the highest or two highest dose levels both in the absence and presence of S9 metabolism.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MAIN ASSAY
A single Main Assay was performed. The maximum concentration of the test item to be used in the main experiment 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 tests, in the Main Assay, using the plate incorporation method, the test item was assayed at the following dose levels:
TA1535 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247, 0.123 μg/plate
TA1535 (with S9): 15.8, 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
WP2 uvrA (without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
WP2 uvrA (with S9): 15.8, 7.90, 3.95, 1.98, 0.988 μg/plate
TA1537 (with and without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate
TA 100 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate
TA100 (with S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
TA98 (with and without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate
Slight toxicity, as indicated by thinning of the background lawn, was observed at the highest or two highest dose levels both in the absence and presence of S9 metabolism with all tester strains, with the exception of WP2 uvrA and TA98.
No precipitation of the test item was observed at the end of the incubation period, at any concentration in the absence or presence of S9 metabolic activation.
The test item induced large and dose related increases in the number of revertant colonies both in the absence and presence of S9 metabolism with all tester strains, with the exception of TA1535. Since a clear positive response was observed, no further experiment was undertaken.
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 4-anilinobenzenediazonium hydrogen sulphate was assayed in the first toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. Severe toxicity, as indicated by microcolony formation or complete lack of bacterial growth, was observed at all dose levels with all tester strains both in the absence and presence of S9 metabolism. In order to exclude a toxic effect related to a marked change in the pH of the medium, 50 µl of a stock solution at 100 mg/ml was added to 0.5 ml of phosphate buffer (pH 7.4,0.1 M) and the pH was measured. The result obtained (6.94) indicated that the pH of treatment mixtures was in the physiological range. A second experiment was performed lowering the concentration range. The test item was assayed at a maximum dose level of 15.8 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 5.00, 1.58, 0.500 and 0.158 µg/plate. Toxicity, from marked to slight, was observed both in the absence and presence of S9 metabolic activation, at the highest or at higher dose levels with all tester strains with the exception of WP2 uvrA in the presence of S9 metabolism. Large and dose related increases were observed up to the highest concentration showing a slight toxicity both in the absence and presence of S9 metabolism with all tester strains, with the exception of TA1535.

ACCEPTANCE CRITERIA AND EVALUATION:
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. The study was accepted as valid.
The test item induced dose related and statistically significant increases in the number of revertant colonies based on a “doubling rate” in the absence and presence of S9 metabolism, with all tester strains with the exception of TA1535.

Applicant's summary and conclusion

Conclusions:

The test item 4-anilinobenzenediazonium hydrogen sulphate induces reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism in all strains with the exception of TA1535.

Executive summary:

The test item 4-anilinobenzenediazonium hydrogen sulphate was examined for the ability to induce gene mutations in tester strains of Salmonela 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 dimethylsulfoxide (DMSO).

Toxicity tests

The test item 4-anilinobenzenediazonium hydrogen sulphate was assayed in the first toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Severe toxicity as indicated by microcolony formation or complete lack of bacterial growth was observed at all dose levels with all tester strain/activation condition combinations. An additional experiment (second toxicity test) was carried out using a lower concentration range. The following dose levels were used: 15.8, 5.00, 1.58, 0.500 and 0.158 µg/plate. Toxicity, from marked to slight, was observed both in the absence and presence of S9 metabolic activation, at the highest or at higher dose levels with all tester strains with the exception of WP2 uvrA in the presence of S9 metabolism. Large and dose related increases in revertant numbers were observed both in the absence and presence of S9 metabolic activation with all tester strains, with the exception of TA1535.

Main Assay

On the basis of toxicity test results, in the Main Assay, using the plate incorporation method, the test item was assayed at the following dose levels:

TA1535 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247, 0.123 μg/plate

TA1535 (with S9): 15.8, 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate

WP2 uvrA (without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate

WP2 uvrA (with S9): 15.8, 7.90, 3.95, 1.98, 0.988 μg/plate

TA1537 (with and without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate

TA 100 (without S9): 3.95, 1.98, 0.988, 0.494, 0.247 μg/plate

TA100 (with S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate

TA98 (with and without S9): 7.90, 3.95, 1.98, 0.988, 0.494 μg/plate

Slight toxicity, as indicated by thinning of the background lawn, was observed both in the absence and presence of S9 metabolism, at the highest or two highest dose levels, with all tester strains with the exception of WP2 uvrA and TA98.

No precipitation of the test item was observed at the end of the incubation period, at any concentration tested.

The test item induced dose related and large increases in the number of revertant colonies with all tester strains with the exception of TA1535, both in the absence and presence of S9 metabolism. Since a clear positive response was observed, no further experiment was undertaken.

Conclusion

It is concluded that the test item 4-anilinobenzenediazonium hydrogen sulphate induces reverse mutation in Salmonella typhimurium and Escherichia coli both in the absence and presence of S9 metabolism, under the reported experimental conditions.