sodium hydrogen (E)-3-((5-chloro-2-hydroxyphenyl)diazenyl)-4,5-dihydroxynaphthalene-2,7-disulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 6 to December 11, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

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

Details on mammalian cell type (if applicable):

TA 98 and TA 1537: dection of frameshift mutations
TA 100 and TA 1535: detection of base-pair substitution mutations
E. coli WP2 uvrA: detection of cross-linking mutations

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of rat liver homogenate and mixture of cofactors

Test concentrations with justification for top dose:

Plate incorporation test: (15) 50, 150, 500, 1500, 5000 µg/plate
Pre-incubation test: 1000, 2000, 3000, 4000, 5000 µg/plate
No cytotoxicity was seen with TA 98 without metabolic activation in standard plate test as well as in pre-incubation test.

Vehicle / solvent:

- Vehicle/solvent used: water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene, 9-aminoacridine hydrochloride monohydrate, 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Genotypes of strains: genotypes of each strain were controlled (plasmid pKM 101 – ampicillin resistance, uvr mutation, rfa mutation, his/trp mutation – spontaneous reversions).
Preparation and using of S9: metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Cofactors (NADP and glucoso-6-phosphate) were dissolved in buffer.
Each plate in all experiments with metabolic activation contained 0.5 ml of buffer with NADP and glucoso-6-phosphate and 30, 50 or 100 µl S9 (the concentration of S9 in the S9 mix was 5.7, 9.1 or 16.7 %). In experiments without metabolic activation only buffer was added to the top agar.
Controls: each experiment included corresponding positive (reference mutagens) and negative controls (untreated control, solvent control). Untreated controls contain no solvent and negative controls contain 0.1 ml of DMSO. All the control numbers were compared with historical ranges of mutant frequencies obtained in our laboratory. The actual numbers were in ranges of the historical numbers.

Test procedure
Mutagenicity experiment I and toxicity test: 100 µl of test substance of required concentration, 100 µl of 16-18 h culture of tester strain of density 108-109 CFUl ml, 0.5 ml relevant buffer and 30 or 100 µl of S9 post mitochondrial fraction (in case of test with metabolic activation) were added to the 2 ml of molten top agar (with trace of histidine or tryptophan) kept in a test tube at 45±3 °C. After shaking the mixture was poured into a minimal glucose agar plate.

Mutagenicity experiment II: 0.5 ml of relevant buffer, 100 µl of the test substance of required concentration, 100 µl of 16-18 h culture of tester strain of density 108-109 CFU/l and 50/100 µl of S9 post mitochondrial fraction were mixed and shaken at 37±1 °C for 30 minutes (preincubation). Then, 2 ml of molten top agar (with trace of histidine or tryptophan) was added and the mixture was poured into a minimal glucose agar plate.
Petri dishes were incubated of 72 h at 37±1 °C, the number of revertant colonies on the plate was counted manually with exception of positive controls, which were counted by an AccuCount 1000.
For an adequate estimate of variation, triplicate plating was used at each dose level. The toxicity test, which serves for finding of optimal concentrations for the mutagenicity test, was performed in strain TA 98 and two Petri dishes were used for every concentration.

Selection of doses/toxicity: test substance is soluble in water up to 26.62 g per litre so a suspension was prepared to reach the highest recommended concentration 5000 µg per 0.1 ml.
For toxicity experiment the highest concentration 5 mg per ml was diluted to the other 5 concentrations in 3-digit places interval. The concentration row was tested for toxicity in strain TA 98 without metabolic activation.
The undiluted test substance occurred in Petri dishes in the two highest concentrations 2500 and 5000 µg per plate.
No toxicity was observed in any dose. The concentration of 5000 µg per plate was used as maximum in the first mutagenicity experiments. The undiluted test substance was expected in the highest concentration only. Further doses were diluted with factor approximately 2-√10.
In mutagenicity experiment I, cytotoxicity occurred in E. coli. No cytotoxicity was observed in the other bacterial strains.
To modify experimental conditions and to increase the sensitivity of the assay, experiment II with metabolic activation was performed with pre-incubation (30 minutes, 37±1 °C, shaking). The same maximum dose was used but single concentrations were increased. Volume of S9 was increased from 30 to 50 µl.
Fresh suspensions/solutions of the test substance were prepared before each experiment. Concentrations of the test substance suspension/solution were dosed in the volume of 0.1 ml per plate. In all experiments, tubes with application forms of the test substance as well as tubes with top agar were shaken before any handling.


In additon to plate incorporation test (exp. I) and pre-incubation test (exp. II), additional experiments were performed.
Experiment in E. coli with metabolic activation without pre-incubation because of 2 toxic concentrations in mutagenicity experiment I, whereas the highest concentration was omitted and next lower concentration in order (15 μg per plate) was tested.
A mutagenicity experiment III in TA 100 with pre-incubation was performed for confirmation/negation of dose-response.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods (2, 3). After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.
An increase is considered as ”biologically relevant“:
- if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
- if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;
A test substance producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
According to OECD guideline 471, the biological relevance of results is the criterion for the interpretation of results, a statistical evaluation of results is not regarded as necessary.

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

standard plate test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

plate incorporation test and pre-incubation test

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

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

Test results 4

Species / strain:

S. typhimurium TA 1537

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

Test results 5

Species / strain:

S. typhimurium TA 98

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

Test results 6

Species / strain:

S. typhimurium TA 100

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:

Most of results obtained in experiments did not show substantial/biologically significant increases in the number of revertants versus solvent controls (Rt/Rc < 2), and no one of these experiments gave evidence of a rising trend in the number of revertants with increasing dose. In case of E.coli higher toxicity was observed than in Salmonella typhimurium strains.
Signs of mutagenicity were observed in cytotoxicity experiments in Salmonella typhimurium TA 98. Mutagenicity was not confirmed in mutagenicity experiments.
In TA100 strain there is a slight mutagenicity observed; anyway the positivity on TA100 (with and without S9) is not statistically significant; Rt/Rc are always within the 2 fold acceptability limit.
Moreover the positivity observed in TA100 is not biologically significant. The average of data “revertants per plate” is always within the data on historical data of laboratory. Even at 3000 µg/plate, where the value Rt/Rc is equal to 2.0 fold, the value of revertants is 174+4, that is included within the data of historical data of laboratory, therefore not to consider significant.
It can be concluded that test substance is not mutagenic for Salmonella typhimurium TA 98, TA100, TA 1535, TA 1537 strains and E. coli WP2 uvrA strain without as well as with metabolic activation.

Applicant's summary and conclusion

Conclusions:

Non mutagenic for TA 1535, TA 1537, TA 98, TA 100 strains and E. coli WP2 uvrA strain with and without metabolic activation.

Executive summary:

Method

Mutagenicity of test substance was assayed in a bacterial reverse mutation test, based on EU method B.13/14 and OECD guideline 471.

Four Salmonella typhimurium strains, TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA strain were used. Test substance was dissolved in water for injection and assayed in doses of (15) 50 - 5000 µg/plate, which were applied to plates in volume of 0.1 ml.

Mutagenicity experiments I were performed without and with metabolic activation using a supernatant of rat liver (S9; 30μl or 100 μl per plate) and a mixture of cofactors by the plate incorporation test with a dose range of 50 – 5000 µg/plate.

To modify experimental conditions and to increase the sensitivity of the assay, mutagenicity experiments II were performed with pre-incubation and dose-range 1000-5000 µg/plate. Volume of S9 was increased to 50 μl per plate.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory.

Results

In mutagenicity experiments, test substance was non-mutagenic for Salmonella typhimurium TA 98, TA100, TA 1535, TA 1537 strains and E. coli WP2 uvrA strain without as well as with metabolic activation.