Reaction products of diazotised Metanilic Acid coupled with 8-Hydroxyquinoline, subsequently metalized with Chromium (III) Basic Sulfate and Sodium Formate, sodium salts

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 09th February 2018 to 05th March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

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:

1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Acid Orange 061 (a)

IUPAC Name:

Acid Orange 061 (a)

Test material form:

solid: particulate/powder

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Standard S9 mix (rat) and Reductive S9 mix-Prival modification (hamster)

Test concentrations with justification for top dose:

-Toxicity test: 5000, 1580, 500, 158 and 50.0 µg/plate; the choice was based on results of the performed solubility test.
-Main Assay I and II: 5000, 2500, 1250, 625, 313 (for all strains); the choice was based on the results obtained in the preliminary toxicity test, taking into consideration cytotoxicity and solubility in the final treatment mixture.

Vehicle / solvent:

Sterile water for injection (Baxter, batches 16E0902 and 17C3006).
This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity and on basis of the solubility test performed

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

congo red

methylmethanesulfonate

other: 2-Aminoanthracene in DMSO and Trypan blue Solution 0.4%

Details on test system and experimental conditions:

BACTERIAL STRAINS
TA1535 and TA100 are predominantly sensitive to base pair mutagens, TA1537 and TA98 are sensitive to frameshift mutagens. In addition to a mutation in the histidine operon, the Salmonella tester strains contain additional mutations which enhance their sensitivity to some mutagenic compounds. The rfa wall mutation results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide barrier that forms the surface of the bacterial cell wall and increases permeability to certain classes of chemicals. All strains are deficient in a DNA excision repair system (uvrB mutation) which enhances the sensitivity to some mutagens. TA98 and TA100 strains contain the pKM101 plasmid which activates an error prone DNA repair system.
Tester strain WP2 uvrA is reverted from tryptophan dependence (auxotrophy) to tryptophan independence (prototrophy) by base substitution mutagens. In addition to the mutation in the tryptophan operon, the tester strain contains a uvrA DNA repair deficiency which enhances its sensitivity to some mutagenic compounds.
Permanent stocks of these strains are kept at -80°C. 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 onMinimal plates+Biotin and Growth onMinimal plates+Biotin+Histidine.
Tryptophan requirement: No Growth onMinimal agar plates and Growth onMinimal plates+Tryptophan.
uvrA, uvrB: Sensitivity to UV irradiation.
rfa: Sensitivity to Crystal Violet.
pKM101 Resistance to Ampicillin.
Bacterial cultures in liquid and on agar were clearly identified with their identity.

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-BonnerMedium 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, 10mL of a sterile solution of 0.5 mM Biotin + 0.5 mM Histidine (or 0.5mMtryptophan) was added to the top agar (100 mL).

METHOD OF APPLICATION:
- Main Assay I: plate-incorporation
- Main Assay II: pre-incubation

SOLUBILITY TEST
Solubility of the test item was evaluated in a preliminary trial using distilled water.

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. Treatment was performed both in the absence and presence of S9 metabolism (RatMixed Induction) 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 and 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.
- Main Assay I
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 0.1 mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL
Bacterial suspension 0.1 mL
- Main Assay II
The components were added in turn to an empty test-tube:
Bacterial suspension 0.1 mL
Test item solution 0.1 mL
(Positive control item solution)* (0.05 mL)
Modified S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5 mL
* For Congo Red and Trypan Blue, 0.1mL was added instead of 0.05 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.
The results presented for TA100 tester strain in the absence and presence of S9 metabolism using the pre-incubation method were obtained in a repeat assay. In the original experiment, the spontaneous mutant frequency of this tester strain fell out the historical control range.

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 andMain Assay II were immediately scored by counting the number of revertant colonies on each plate, while plates
from Main Assay I were held at 4 °C for approximately 24 hours before scoring.

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 fited 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).
Regression lines were 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 were also given.

Results and discussion

Test results

Species / strain:

other: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

SOLUBILITY
The test item was found to be soluble at 50.0 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

TOXICITY TEST
No precipitation of the test item was observed at the end of the incubation period, at any concentration.
Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.
Plates treated with the test item presented a dose dependent orange colour of the agar which did not interfere with the scoring.

MAIN ASSAYS
-Main Assay I: no toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation and no relevant increase in revertant numbers was observed at any concentration tested.
-Main Assay II: no toxicity was observed at any concentration tested, with any tester strain, in the absence or presence of S9 metabolic activation. Slight increases in revertant numbers were observed in the presence of S9 metabolism with TA100 tester strain reaching 1.9-fold the concurrent negative control value at the intermediate dose level of 1250 µg/plate. No increases were observed with any of the remaining tester strain/activation condition combination. No precipitation of the test item, evident to the unaided eye, was observed at the end of the incubation period at any concentration, in any experiment, in the absence or presence of S9 metabolism.

In both assays, plates treated with the test item presented a dose dependent orange colour of the agar (orange-yellow in the presence of the reductive metabolic system) which did not
interfere with the scoring of colonies.
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.

EVALUATION CRITERIA
The test item did not induce increases in the number of revertant colonies which were twofold greater than the control values at any dose level, in any tester strain, in the absence or presence of S9 metabolism. On the basis of the stated criteria, it must be concluded that the test item is not mutagenic in bacteria, under the reported experimental conditions.

Remarks on result:

other: Main Assay I

Any other information on results incl. tables

ACCEPTANCE CRITERIA

The study was accepted as valid.

1. Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

The following exceptions occurred in Main Assay II:

– the positive control value for TA1537, in the presence of S9 metabolism, was slightly higher than the upper limit of historical control range. Since response to the positive control was unequivocal, the experiment with this tester strain was deemed acceptable.

2. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain.

3. No plates were lost through contamination or cracking.

Applicant's summary and conclusion

Conclusions:

The test item did not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Executive summary:

The test item was examined for the ability to induce gene mutations in five tester strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100) and Escherichia coli (WP2 uvrA), as measured by reversion of auxotrophic strains to prototrophy according to the OECD 471 (1997). 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 (standard metabolic activation) in Main Assay I, and liver S9 fraction from uninduced hamsters (reductive metabolic activation system with Prival modification method), in Main Assay II. The test item was used as a solution in sterile water for injection.

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals.

No precipitation of the test item was observed at the end of the incubation period at any concentration. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

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 5000, 2500, 1250, 625 and 313 µg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, Main Assay II was carried out using the same concentrations of Main Assay I.

Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

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 any metabolic activation system.

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.