Disodium 3,3'-[[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-2,4-diyl]bis[imino(3-methoxy-4,1-phenylene)azo]]bis[benzenesulphonate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No genotoxicity

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From November 16, to December 13, 2017

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach has been detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July 2016

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Supplier: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany.
- Storage: cells were stored in liquid nitrogen in the cell bank oftesting facility, allowing the repeated use of the same cell culture batch in experiments.
- Stock cultures: thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks.
- Seeding: about 2-3×10^6 cells were seeded into each flask with 15 ml of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/ml) and amphotericin B (1 %).
- Subculturing: the cells were sub-cultured once or twice weekly.
- Incubation: at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.

PERIODICAL CHECKS
- Periodically checked for Mycoplasma contamination: each master cell stock is screened for mycoplasm contamination.
- Periodically checked for karyotype stability: each master cell stock is checked for karyotype stability.
- Periodically 'cleansed' against high spontaneous background: each master cell stock is checked for spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal fraction S9 mix

Test concentrations with justification for top dose:

- Main test: without S9 mix 72.6; 145.2, 290.4, 580.8 and 1161.5 μg/ml; with S9 mix 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml.
- Toxicity data, pre-experiment: 18.1, 36.3, 72.6; 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml
- Selection for main test: the concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment.
- Spacing factor: the individual concentrations were spaced by a factor of 2.

Vehicle / solvent:

- Vehocle: on the day of the experiment (immediately before treatment), the test item was dissolved in deionised water.
- Concentratin: the final concentration of deionised water in the culture medium was 10 %.
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
- Formulation preparation: all formulations were prepared freshly before treatment and used within two hours of preparation.

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

TREATMENT
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 μl/ml S9 mix. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with PBS.
Immediately after the end of treatment the cells were trypsinised as described above and sub-cultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 ml medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival (cloning efficiency I) as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2.
The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment.
Three or four days after first sub-cultivation approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 ml medium.
Following the expression time of 7 days five 75 cm² cell culture flasks were seeded with about 4 to 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability (cloning efficiency II).
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

SEEDING
Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline.
Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

CULTURE MEDIUM
- Seeding: MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/ml), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium.
- Mutant selection: for the selection of mutant cells the complete medium was supplemented with 11 μg/ml 6-thioguanine.
- Incubation: all cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

PRE TEST ON TOXICITY
The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
Approximately 1.5 million cells were seeded in 25 cm² flasks 24 hours prior to treatment. After approximately 24 hours the test item was added and the treatment proceeds for 4 hours (duplicate cultures per concentration level). Immediately after treatment the test item was removed by rinsing with PBS. Subsequently, the cells were trypsinized and suspended in complete culture medium. After an appropriate dilution the cell density was determined with a cell counter. Toxicity of the test item is evident as a reduction of the cell density compared to a corresponding solvent control. A cell density of approximately 1.5 million cells in 25 cm² flasks is about the same as approximately 10 million cells seeded in 175 cm² bottles 24 hours prior to treatment with the main experiment.

OSMOLARITY AND pH
The osmolarity and the pH-value were determined in culture medium of the solvent control and at the maximum concentration of the pre-experiment without metabolic activation.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Metabolic activation system: phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system. T
- S9 capability for activation: each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.
- S9 use: an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/ml in the cultures.
- S9 mix composition: MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).
- Protein concentration: 32.7 mg/ml in the pre-experiment and the main experiment.

ACCEPTABILITY OF THE ASSAY
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
d) An adequate number of cells and concentrations (at least four test item concentrations) are analysable even for the cultures treated at concentrations that cause 90 % cytotoxicity during treatment.
e) The criteria for the selection of the top concentration are fulfilled.

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95 % control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95 % control limits).
In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data should be evaluated by expert judgement or further investigations.

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

Precipitation visible at the end of treatment was noted at 1161.5 μg/ml without metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.
No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations. The range of the mean mutant frequencies of the groups treated with the test item was from 5.4 up to 27.6 mutants per 10^6 cells. The linear regression analysis showed no significant dose dependend trend of the mutation frequency at any of the experimental groups.

CONTROLS
The mean mutant frequency obtained in the solvent controls was 12.3 without S9 mix and 22.4 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476.
EMS (300 μg/ml) and DMBA (2.3 μg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

PRE TEST ON TOXICITY
In the pre-experiment no toxic effects were observed after 4 hours in the presence and absence of metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 2323.0 μg/mL without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

OSMOLARITY AND pH
Osmolarity: solvent 291 mOsm; test item 299 mOsm.
pH: solvent 7.40; test item 7.39.

Conclusions:

The substance is considered to be non-mutagenic in the HPRT assay perfromed.

Executive summary:

The study was performed to investigate the potential of test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The main experiment was performed with a treatment period of 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment (2323 μg/ml) was chosen with respect to the OECD guideline 476 regarding the purity of the test item. The following concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment: 72.6, 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml.

Precipitation visible at the end of treatment was noted at 1161.5 μg/ml without metabolic activation. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.

No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations.

The mean mutant frequency obtained in the solvent controls was 12.3 without S9 mix and 22.4 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 5.4 up to 27.6 mutants per 10⁶ cells.

The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusion

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the substance is considered to be non-mutagenic in this HPRT assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From May 17 to June 05, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach has been detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted on May 26, 1983

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELL CHECK
Regular checking of the properties of the Salmonella strains with regard to membrane permeability and ampicillin resistance as well as normal spontaneous mutation rates is performed in testing laboratory, according to Ames et al. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

STORAGE
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

hamster liver microsomal fraction

Test concentrations with justification for top dose:

10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate
According to the results of the pre-experiment the concentrations applied in the main experiments are chosen.

Vehicle / solvent:

- Vehicle: bidest. water.
- Justification for choice of vehicle: the vehicle was chosen to its solubility properties and its non-toxicity for the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

congo red

other: 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubation.
The following materials were mixed in a test tube and gently shaken at 30 °C for 30 min.: 100 µl test solution at each dose level, solvent control, negative control, or reference mutagen solution (positive control); 500 µl S9 mix (for test with metabolic activation) or S9 mix substitution-buffer (for test without metabolic activation); 100 µl Bacteria suspension (cf. test system, pre-culture of the strains).

After pre-incubation 2.0 ml of molten 45 °C overlay agar was added to each tube. The mixture was poured on minimal agar plates.
After solidification the plates were incubated upside down for 72 hours at 37 °C in the dark.

NUMBER OF REPLICATIONS: the assay was performed in two independent experiments, using identical procedures. Each concentration, including the controls, was tested in triplicate.

CONDITIONS
- Precultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium.
- Nutrient medium: the nutrient medium contains per litre 8 g Difco Nutrient Broth and 5 g NaCl. - Incubation: the bacterial culture was incubated in a shaking water bath for 6 hours at 37 °C.
- Selective Agar: 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used.
- Dished preparation: each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121 °C in an autoclave.
- Overlay Agar: overlay agar contains per litre 6.0 g Difco Bacto Agar, 6.0 g NaCl, 10.5 mg L-histidine x HCl x H2O and 12.2 mg biotin. Sterilizations were performed at 121° C in an autoclave.

PRE-EXPERIMENT FOR TOXICITY
To evaluate the toxicity of the test article a prestudy is performed with strains TA 98 and TA 100. 8 concentrations are tested for toxicity and mutation induction with each 3 plates.
The experimental conditions in the pre-experiment are the same as described for the main experiments.
Toxicity of the test article may be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial

DATA RECORDING
The colonies were counted using the BIOTRAN III counter. The counter was connected to an IBM XT compatible PC with printer which printed out the individual values and the means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates. If precipitation of the test article precluded automatic counting the revertant colonies were counted by hand.

MAMMALIAN MICROSOMAL FRACTION S9 MIX
The S9 liver microsomal fraction was obtained from the liver of 7 - 8 weeks old male Syrian golden hamsters. After cervical dislocation the livers of the animals were removed, washed in 0.1 M sodium phosphat buffer pH 7.4, 0.25 M sucrose and 1 mM disodium EDTA in bidestilled water and homogenised. The homogenate, diluted 1:3 in sodium phosphat buffer was centrifuged cold at 9000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2 or 5 ml and stored at -70 °C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.

S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution in a ratio 3:7.
The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix: 33 mM KCl, 8 mM MgCl2, 20 mM glucose 6-phosphate, 2.8 units/ml glucose 6-phosphate dehydrogenase, 4 mM NADP, 2 NADH, 2 FMN in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The preparation was performed according to Ames et al. and and Mitchell.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates.
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

weak toxic effects

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).
The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.
The weak increases of revertant colony numbers in the strain TA 1535 (exp. I, without S9 mix at 10.0 and 1000.0 µg/plate), in TA 98 (exp. I, with S9 mix at 10.0 µg/plate) and in TA 1537 (exp. II, with S9 mix at 333.3 µg/plate) are considered not to be relevant. The obtained effects were not reproduced in the independent experiment.

CONTROLS
Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

PRE-EXPERIMENT FOR TOXICITY
The plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.

Conclusions:

The substance is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay performed.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations according to the pre-incubation test for azo dyes using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100.

The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate.

Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I).

The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used.

The test article induced a slight increase in the number of revertants in strain TA 1535 at 10.0 and 1000.0 µg/plate without metabolic activation (exp. I), in TA 98 at 10.0 µg/plate (exp. I) and in TA 1537 at 333.3 µg/plate (exp. II) both with metabolic activation.

Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the Salmonella typhimurium strains used. The presence of liver microsomal activation did not influence these findings.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusion

The substance is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay performed.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

November from 14 to 25, 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach has been detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

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

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr. Bruce N. Ames, University of California at Berkeley, U.S.A. (1987).
- Storage: stock cultures of the four strains were stored in liquid nitrogen (-196 °C).
- Periodically check: strains were regularly checked for their histidine-requirement, crystal vioiet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

PREPARATION OF BACTERIAL CULTURES
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid no. 2) and incubated in a shaking bath (37 °C, 150 spm), until the cultures reached an O.D. of 0.4 at 700 nm (10^9 cells/ml). Freshly grown cultures of each strain were used for a test.

Metabolic activation:

with and without

Metabolic activation system:

rat liver microsomal enzymes

Test concentrations with justification for top dose:

Main experiments: 100, 333, 1000, 3330 and 5000 µg/plate.
Preliminary toxicity test: 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330 and 5000 µg/plate.
Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix.

Vehicle / solvent:

- Solvent: the test substance has been dissoived in dimethysulphoxide of spectroscopic quafity.
- Preparation of solution: test substance concentrations were prepared directly prior to use.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycine // 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation). Top agar in top agar tubes was melted and heated to 45 °C. The following solutions were successively added to 3 ml top agar: 0.1 ml of a fresh bacterial culture (10^ cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test substance in dimethylsulphoxide of spectroscopic quality and either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were turned and incubated in the dark at 37 °C for 48 h.
- Agar plate: agar plates (Ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid, code L28) in Vogel-Bonner Medium E, 10 g glucose, 0.5 mg biotin and 0.6 mg histidine.
- Top Agar: Vogel-Bonner Medium E containing 0.6 % (w/v) purified agar was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 120 °C. Prior to the viability determination the top agars were supplemented with 1.55 mg histidine per top agar.

INCUBATION CONDITIONS: all incubations have been carried out in the dark at 37 °C. The temperature was monitored during the experiment.

NUMBER OF REPLICATIONS: 5 different doses (with approximately half-log steps) of the test substance have been tested in triplicate in each strain.

COLONY COUNT: the revertant colonies (histidine independent) have been counted automatically with an Artek model 880 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting have been counted manually.

PREPARATION OF S-9 HOMOGENATE
Rat liver microsomal enzymes were routinely prepared from adult male Wistar or Sprague Dawley rats, which were obtained from Charles River Wiga, Sulzfeld, F.R.G. The animals were housed at testing laboratory in a special room under standard laboratory conditions. The rats were injected intraperitoneally with a solution (20 % w/v) of Aroclor 1254 (500 mg/kg body weight) in corn oil. Five days later, they were killed by decapitation; (they were denied access to food for at least 12 hours preceding sacrifice). The livers of the rats were removed aseptically, and washed in cold (0 °C) sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. Subsequently the livers were minced in a blender and homogenized in 3 volumes of phosphate buffer with a Potter homogenizer. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196 °C).

Preparation of S9 mix: S9-mix was prepared immediately before use and kept on ice during the test. S9-mix contained per 10 ml: 30 mg NADP and 15.2 mg glucose-6-phosphate in 5.5 ml aqua bidest; 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution; 1 ml 0.33 M KCl solution, and 0.5 ml S9. The solutions were mixed and filter (0.22 um)-sterilized (apart from the S9-fraction, which was added after filter-sterilization of the S9-mix components).

DOSE SELECTION
Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates. If no toxicity was observed, the highest dose level used in the mutagenesis assay was 5 mg/plate unless the test article exhibited limited solubility or was not uniformly dispersible in the solvent of choice.

ACCEPTABILITY OF THE ASSAY
An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably Fall within the laboratory background historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance.
Furthermore, the mean plate count should be at least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding test with strain TA100 or should extend to 5 mg/plate.

Evaluation criteria:

A test substance was considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance was considered positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98 and 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:

All bacterial strains showed negative responses over the entire dose range of the test substance, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values Fell within our laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

TOXICITY OF THE TEST SUBSTANCE
The survival of the TA100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate.
Both in the absence and presence of S9-mix the survival of strain TA100 is not reduced up to test substance concentrations of 5000 µg/plate. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix.

Conclusions:

The test substance can be considered as not mutagenic in the.test system.

Executive summary:

The substance was tested in the Salmonella/microsome plate test up to 5000 µg/plate in the absence and presence of S9-mix; S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 strains were tested. Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA 100, both with and without S9-mix. In the main experiment 5 different doses (with approximately half-log steps) of the test substance have been tested in triplicate in each strain. The test substance has been tested both with and without S9-mix in each strain, in two independent experiments. The following concentrations were assayed: 100, 333, 1000, 3330 and 5000 µg/plate.

The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains. These results were confirmed in an independently repeated experiment.

The negative and strain-specific positive control values fell within testing laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

In the toxicity test, the survival of the TA 100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. Both in the absence and presence of S9-mix the survival of strain TA 100 is not reduced up to test substance concentrations of 5000 µg/plate. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix.

Conclusion

The test substance can, therefore, be considered as not mutagenic in the.test system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No genotoxicity

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From November 21, 1988 to January 13, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

The read across approach has been detailed in the report attached to the IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, FRG.
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: 20-29 g females; 26-34 g males.
- Fasting period before study: feed was withheld overnight prior to dosing until approximately 3-4 hours after administration of the test substance.
- Housing: in groups of 5 per sex in polycarbonate cages.
- Diet: standard laboratory animal diet RMH-B, pellet diameter 10 mm, Hope Farms, Woerden, The Netherlands. The feed was analysed for contaminants by the manufacturer.
- Water: tap water, ad libitum.
- Acclimation period: at least 6 days under laboratory conditions.
- Health check: on arrival and prior to final assignment to study, all animals have undergone a detailed clinical examination to ensure selected animals were in a good state of health.

ENVIRONMENTAL CONDITIONS
- Temperature:21± 3 °C
- Relative humidity: 40 - 70 %
- Air changes: air-conditioned room, 7.5 air changes per hour.
- Photoperiod: 12 hours artificiai fiuorescent iight/12 hours dark.

Route of administration:

oral: gavage

Vehicle:

The test substance was dissolved or suspended in Milli-RO water (Millipore Corp., Bedford, Mass., USA).
Test substance concentrations were prepared directiy prior to use.

Details on exposure:

- Dosing volume: 10 ml/kg bw
- Rationale for dose selection: it is generally recommended to use the maximum tolerated dose for the micronucleus assay.

Frequency of treatment:

Single dosing

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 male and 5 female mice per treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Erythrocytes in mouse bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Selection of an adequate dose for the Micronucleus test was based on a pilot study. Depending on the available toxicity data 1 to 5 dose groups, each comprising 3 males and 3 females, received a single dose of the test substance. Generally, the study duration is 3 days, however this might be changed if considered necessary. During this period mortality and physical condition has been recorded daily.

TREATMENT AND TISSUES PREPARATION
- Sacrifice: the animals were sacrificed by cervical disiocation at 24, 48 and 72 hours after dosing of the test substance and the vehicie and at 48 hours after dosing of the positive control.
- Cells suspension prepoaration: both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed by aspiration with the serum.

DETAILS OF SLIDE PREPARATION
- Slides preparation: a drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96 % ethanol/ether and cleaned with a tissue) and marked. The drop was spread by moving a clean slide with round-whetted sides at an angle of 45 °C over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100 % methanol and air-dried overnight.
- Replicates: two slides were prepared per animal.
- Staining of the bone marrow smears: the slides were stained for 3 min in undiluted May-Grünwald solution followed by 2 min in May-Grünwald solution diluted 1:1 with Sdrensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5 % (v/v) Giemsa solution in Sdrensen buffer pH 6.8. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

METHOD OF ANALYSIS
At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Averages and standard deviations were calculated.

ACCEPTABILITY OF ASSAY
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.

Evaluation criteria:

A test substance is considered positive in the micronucieus test if:
a) It induced a bioiogically as well as a statistically significant (wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucieus test if:
a) None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.
The preceding criteria are not absolute and other extenuating factors may enter into the final evaluation decision.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mean number of micronuclei scored in the test substance-treated groups was compared with the corresponding control groups. No increase in the frequency of micronuclei was observed.

CONTROLS
The incidence of micronuclei in the control animals was found to be in the range of historical data (0.75 1 0.97; mean 3 standard deviation, N = 700).
The groups that were treated with Cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis. The positive control substance induced in both sexes a statistically significant increase in the number of micronuclei.

DOSE SELECTION
In a preliminary study 12 animals (3 males and 3 females per group) were dosed orally with 2000 and 1000 mg/kg body weight (groups A and B, respectively).
Higher concentrations could not be dosed because of aggregation of the test substance in suspension. Animals of group A and B did not show any signs of reaction to treatment.
Based on the results of this pilot study 2000 mg/kg body weight was selected as an appropriate dose for the Micronucleus Test.

Conclusions:

Test item can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions described in the report.

Executive summary:

The substance was tested in the Micronucleus Test in mice. Three groups, each comprising 5 males and 5 females, received a single oral dose of 2000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

Conclusion

It is concluded that test item can be considered as not mutagenic in the Mouse Micronucleus Test under the experimental conditions described in the report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the in vitro AMES assay performed on Similar substance 01, in vitro gene mutation on mammalian cells performed on the structural analogous Similar Substance 02 and in vivo chromosomal aberration conducted on Similar Substance 01.

The read across approach can be considered as reliable and suitable for the purpose; details and explanations are detailed in the report attached to the IUCLID section 13.

IN VITRO GENE MUTATION IN BACTERIA

A study was performed to investigate the potential of the Similar Substance 01 to induce gene mutations according to the pre-incubation test for azo dyes using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100. The test article was tested at the following concentrations: 10.0, 100.0, 333.3, 1000.0 and 5000.0 µg/plate. Only weak toxic effects, evidenced by a reduction in the number of spontaneous revertants, occurred at the highest investigated dose in TA 1537 with metabolic activation (exp. I) and in TA 98 at 10.0 and 5000.0 µg/plate without metabolic activation and at 5000.0 µg/plate with metabolic activation (exp. I). The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. The test article induced a slight increase in the number of revertants in strain TA 1535 at 10.0 and 1000.0 µg/plate without metabolic activation (exp. I), in TA 98 at 10.0 µg/plate (exp. I) and in TA 1537 at 333.3 µg/plate (exp. II) both with metabolic activation.

Up to the highest investigated dose, no significant and reproducible dose-dependent increase in revertant colony numbers was obtained in any of the strains used. The presence of liver microsomal activation did not influence these findings.

A second test was conducted in the same Salmonella typhimurium strains, i.e. TA 1535, TA 1537, TA 98 and TA 100. Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. In the main experiment 5 different doses were assayed: 100, 333, 1000, 3330 and 5000 µg/plate. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains. These results were confirmed in an independently repeated experiment. In the toxicity test, the survival of the TA 100 culture is determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. Both in the absence and presence of S9-mix the survival of strain TA 100 is not reduced up to test substance concentrations of 5000 µg/plate. Based on these data, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix.

IN VITRO GENE MUTATION IN MAMMALIAN CELLS

The study was performed to investigate the potential of Similar Substance 02 to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The main experiment was performed with a treatment period of 4 hours with and without metabolic activation. The following concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment: 72.6, 145.2, 290.4, 580.8, 1161.5 and 2323.0 μg/ml.

Precipitation visible at the end of treatment was noted at 1161.5 μg/ml without metabolic activation. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % was observed neither in absence nor presence of metabolic activation.

No biologically relevant increase in mutant colony numbers was observed in the main experiment up to the maximum concentrations scored for gene mutations.

The mean mutant frequency obtained in the solvent controls was 12.3 without S9 mix and 22.4 with S9 mix. The values were well within the 95 % confidence interval of our laboratory’s historical negative control data and, thus, fulfilled the requirements of the current OECD Guideline 476. The range of the mean mutant frequencies of the groups treated with the test item was from 5.4 up to 27.6 mutants per 10⁶ cells. The linear regression analysis showed no significant dose dependent trend of the mutation frequency at any of the experimental groups.

EMS and DMBA were used as positive controls and showed a distinct increase in induced mutant colonies.

Under the experimental conditions reported, it was concluded that the test item did not induce gene mutations at the HPRT locus in V79 cells.

IN VIVO CHROMOSOMAL ABERRATION

Similar Substance 01 was tested in the micronucleus test in mice. Three groups, each comprising 5 males and 5 females, received a single oral dose of 2000 mg/kg body weight. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide at 50 mg/kg body weight, was harvested at 48 hours after dosing only. The test substance was found to respond negatively in the Micronucleus Test, whereas the positive control substance produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes. Therefore, the test item was judged as not mutagenic in the Mouse Micronucleus Test under the experimental conditions described.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the criteria to classify the substance for genetic toxicity are not met, according to the CLP Regulation (EC) No 1272/2008.