Trisodium bis[4-[4,5-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzene-1-sulphonato(3-)]cobaltate(3-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (OECD 471): positive

Chromosome aberration assay (OECD 473): positive

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29th to 31st October, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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:

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).

Rat liver S9 fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was used.

S9 mix (with rat liver S9)
Salt solution for S9 mix Final concentration in S9 mix
NADP Na 7.66 g 4 mM
D-glucose-6 phosphate Na 3.53 g 5 mM
MgCl2 1.90 g 8 mM
KCl 6.15 g 33 mM
Ultrapure water ad 1000 ml
Sterilized by filtration through a 0.22 µm membrane filter.

The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate-buffer, pH 7.4 500 ml
Rat liver homogenate (S9) 100 ml
Salt solution for S9 mix 400 ml
The S9 mix (containing 10 % S9) was kept in an ice bath before it was added to the culture medium.

The present test item contains azo-bonds. Therefore, the modified protocol proposed by Prival and Mitchell [10] (e.g.: using of hamster liver S9 Mix that contains 30 % hamster liver extract etc.) was originally planned for the confirmatory mutation test.
However, in this study unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test); therefore, further investigation, following the modified protocol proposed by Prival and Mitchell was considered as not necessary for the final conclusion of the study.

Sodium phosphate buffer (0.2 M, pH 7.4)
Solution A:
Na2HPO4 × 12H2O 71.63 g
Ultrapure water ad 1000 ml
Solution B:
NaH2PO4 × H2O 27.6 g
Ultrapure water ad 1000 ml
Solution A 880 ml
Solution B 120 ml*
* The components were mixed in the above ratio; thereafter the pH was checked and corrected. The correction was performed with admixture of the solution A or B.
After the pH setting the sterilization was performed by filtration through a 0.22 µm membrane filter.

Test concentrations with justification for top dose:

Concentration tested in µg/plate: 5000, 1600, 500, 160, 50, 16, 5
Justification of concentrations: selection of the concentrations for the initial mutation test was based on the results of the solubility trials and the concentration range finding test (informatory toxicity test).
The maximum test concentration was 5000 µg/plate (±S9), as recommended in the guideline. For the dose selection the cytotoxicity, the solubility and a possible positive effect of the test item were taken into consideration.
In the main test, at the preparation of the test item stock solution a correction (multiplier) factor of 1.25 (1/0.80=1.25) based on its purity of 80 % was taken into consideration.
For facilitating the test item dissolution, in the main test, for the preparation of test item stock solution 2-3 min. ultrasonic treatment was applied.
The test solutions were freshly prepared at the beginning of the experiment.

Vehicle / solvent:

- DMSO for test item
- DMSO or ultrapure water for positive controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Genotypes of the strains used for mutagenicity testing
In addition to mutations in genes involved in histidine or tryptophan synthesis, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Storage of tester strains
The strains are stored at -80 ± 10 °C. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of phenotypes of tester strains
The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al..

Spontaneous reversion of tester strains
Each tester strain reverts spontaneously at a frequency that strain specific. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate. The historical control values for spontaneous revertants are available.

Procedure for bacterial cultures
The frozen bacterial cultures were thawed at room temperature and 200 µl inoculum was used to inoculate each 50 ml of Nutrient Broth No. 2 for the overnight cultures in the assay. The cultures were incubated for approximately 10-12 hours in a 37 °C Benchtop Incubator Shaker.

Viability and the cell count of the testing bacterial cultures
Fresh cultures of bacteria were grown up to the late exponential or early stationary phase of growth (approximately 10^9 cells per ml). Cultures in late stationary phase were not used. The titer was demonstrated in each assay through the determination of viable cell numbers by a plating experiment.
The viability of each testing culture was determined by plating 0.1 ml of the 10^-5, 10^-6, 10^-7 and 10^-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Media
An appropriate minimal agar and an overlay agar containing histidine and biotin or tryptophan, to allow for a few cell divisions were used.

The Minimal Glucose Agar (MGA) plates
Ready-to-use minimal glucose agar (MGA) plates were used in the study.
Typical composition (g/1000 ml) of MGA plates:
Glucose 20.0 g
Magnesium sulfate 0.2 g
Citric acid 2.0 g
di-Potassium hydrogenphosphate 10.0 g
Sodium ammonium hydrogenphosphate 3.5 g
Agar agar 13.0 g

Nutrient broth no. 2
Nutrient broth no. 2 25.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Nutrient agar
Nutrient agar 20.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Top agar for Salmonella typhimurium strains
Agar solution:
Agar bacteriological 4.0 g
NaCl 5.0 g
Ultrapure water ad 1000.0 ml
Sterilization for 20 minutes was performed at 121˚C in an autoclave.

Histidine – biotin solution (0.5 mM):
D-Biotin 122.2 mg
L-Histidine•HCl H2O 104.8 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete top agar for Salmonella typhimurium strains:
Histidine – Biotin solution (0.5 mM) 100.0 ml
Agar solution 900.0 ml

Top agar for Escherichia coli strain
Tryptophan solution (2 mg/ml):
L-Tryptophan 2000.0 mg
Ultrapure water ad 1000.0 ml
Sterilization was performed by filtration through a 0.22 µm membrane filter.

Complete top agar for Escherichia coli strain:
Nutrient broth 50.0 ml
Tryptophan solution (2 mg/ml) 2.5 ml
Agar solution 947.5 ml

Description of test procedure
The study included a preliminary solubility test, a preliminary concentration range finding test (informatory toxicity test) and an initial mutation test (plate incorporation test).
In the preliminary concentration range finding test as well as in the initial mutation test the plate incorporation method was used. Because of the unequivocal, positive results of the initial mutation test further testing for the final conclusion was considered as not necessary.

Solubility test
In the solubility trials the test item behavior was investigated in the applied test system when formulated in dimethyl sulfoxide (DMSO). The test item was dissolved in dimethyl sulfoxide (DMSO). The obtained solutions with the solution of top agar and phosphate buffer were examined in a test tube without test bacterium suspension.

Concentration of test item in the solvent (mg/ml): 50
Solubility, visible behavior in the solvent: clear solution
Solubility in the top solution (final treatment mixture) (test item solution 100 µl + phosphate buffer 500 µl + top agar 2 ml): clear solution
Test item concentration in the test tube µg/tube: 5000

In the solubility test any correction factor, based on the purity of the test item (80 %) was not taken into consideration; therefore the 50 mg/ml solution concentrations corresponded to 40 mg active component/ml.

Concentration range finding tests (informatory toxicity tests)
Based on the solubility trials, the stock solution with a concentration of 50 mg/ml (40 mg/ml active ingredient content) was prepared in DMSO and diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 4000, 1280, 400, 128, 40, 13 and 4 µg active ingredient content/plate, (planned concentrations: 5000, 1600, 500, 160, 50, 16 and 5 µg test item/plate).
In the informatory toxicity test a correction factor, based on the purity of the test item (80 %) was not taken into consideration.
In the performed informatory toxicity test inhibitory effect of the test item was not observed, the colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Biologically relevant dose-related changed revertant colony number increases, high revertant colony numbers above the corresponding genotoxicological thresholds for being positive were obtained in both strains at 4000-400 µg/plate in the absence of exogenous metabolic activation ( S9); in S. typhimurium TA98 down to and including the concentration of 40 µg/plate in the presence of exogenous metabolic activation (+S9), in TA100 at 4000, 1280 and 128 µg/plate (+S9).
The high revertant colony numbers were above the corresponding historical control data range in S. typhimurium TA98 at 13 µg/plate (+S9), in TA100 at 400, 40 and 13 µg/plate (+S9).
No precipitation of the test item was observed on the plates in the above bacterial strains at any examined concentration level (±S9).

Procedure for the initial mutation test
A standard plate incorporation procedure was performed as an initial mutation test. Bacteria were exposed to the test item both in the presence and absence of rat liver S9 as metabolic activation system. Molten top agar was prepared and kept at 45 °C. Two ml of top agar was aliquoted into individual test tubes (3 tubes per both control and each concentration level). The equivalent number of minimal glucose agar plates was properly labelled. Conditions were investigated in triplicate. The test item and other components were prepared fresh and added to the overlay (45 °C).
The content of the tubes:
top agar 2000 µl
solvent or solution of test item or positive controls 100 µl
overnight culture of test strain* (containing approximately 10^8 viable cells) 100 µl
phosphate buffer (pH: 7.4) or S9 mix 500 µl
* : Salmonella typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA.
This solution was mixed and poured on the surface of the properly labeled minimal agar plates (3 plates per both control and each concentration level). For incubations with metabolic activation, instead of phosphate buffer, 0.5 ml of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions and each with the addition of negative and positive controls. The plates were incubated at 37 °C for about 48 hours.
Unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test) in the Salmonella typhimurium TA98, TA100 and TA1537 strains (indicator strains for base-pair substitution and frameshift mutations), in the absence and also in the presence of exogenous metabolic activation (±S9); therefore, the performance of an additional confirmatory experiment was considered as not necessary for the final conclusion of the study.

Evaluation criteria:

The evaluation of revertant colony count, background lawn development and test item precipitate were performed manually by unaided eye.
The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined, the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate (MR) = Mean number of revertants at the test item (or control*) treatments / mean number of revertants of solvent control
* : untreated, solvent or positive control

Evaluation and interpretation of results
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the solvent control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the solvent control.

Statistics:

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a negative response: a test article is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Conditions for the validity of the test
The tests are considered valid if:
- all of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- S. typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- E. coli WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the solvent controls.
- tester strain culture titers are in the 10^9 cells/ml order.
- batch of S9 used in this study shows the appropriate biological activity.
- positive control reference items (known mutagens) show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective solvent control, and the number of induced revertant colonies are in accordance with the corresponding historical control ranges.
- there are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

A dose level is considered toxic if
- reduced revertant colony numbers are observed as compared to the mean solvent control value and the reduction shows a dose-dependent relationship, and / or
- the reduced revertant colony numbers are below the historical control data range, and / or
- pinpoint colonies appear, and / or
- reduced background lawn development occurs.

Species / strain:

S. typhimurium, other: TA100, TA98, TA1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 1535 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Validity of the performed experiments
Tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges and showed the adequate strain culture titer.
Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system.
The positive control reference items (diagnostic mutagens) induced the expected, biological relevant increases (more than 3-fold increase) in revertant colonies and most of the number of revertant colonies was within the historical control data range per strain, thereby meeting the criteria for a valid positive control in all experimental phases and all tester strains.
The numbers of revertant colonies in the solvent control (dimethyl sulfoxide) showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in all experimental phases.
Seven concentration levels were investigated in the informatory toxicity test and in the main mutation experiment (initial mutation test).
In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain.
All criteria for the validity of the performed experiments have thus been met.

Controls
In the performed initial mutation test multiple test items were tested with reference values from the common parallel controls.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers in line with the actual historical control data ranges in all strains.
Each of the investigated reference mutagens (positive controls) showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective solvent control in the initial mutation test and additionally the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all tester strains.
The revertant colony numbers of the untreated and ultrapure water control plates in the initial mutation test were slightly higher or lower than the dimethyl sulfoxide (DMSO) control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges.
In summary, the actual values of untreated, solvent and positive controls were in line with the criteria for validity of the assay.

Initial mutation test (plate incorporation test)
In the initial mutation test biologically relevant increases were observed in revertant colony numbers of Salmonella typhimurium TA98, TA100 and TA1537 strains (indicator strains for base-pair substitution and frameshift mutations), in the absence and also in the presence of exogenous metabolic activation (±S9).
The relevant revertant colony number increases, high revertant colony numbers above the corresponding genotoxicological thresholds for being positive were obtained in S. typhimurium TA98 in the concentration range of 5000-500 µg/plate, in the absence and also in presence of exogenous metabolic activation (±S9); in TA100 at the concentration range of 5000-160 µg/plate ( S9) and at 5000 and 1600 µg/plate (+S9); in TA1537 at the concentration range of 5000-160 µg/plate (±S9). In these strains the highest MR values were obtained at the highest examined concentration of 5000 µg/plate and the further revertant colony number increases followed clear dose-relationship (±S9).
Furthermore, the obtained revertant colony numbers were above the corresponding historical control data range, but remained below the genotoxicological threshold for being positive in TA98 at 160 µg/plate (±S9), at 50 µg/plate (+S9) and in TA100 at 160 µg/plate (+S9).
Additionally, the obtained increased revertant colony numbers showed clear dose related tendency, and at 5000 µg/plate (at the highest examined concentration level) were above the corresponding historical control data range, but remained below the genotoxicological threshold for being positive in Salmonella typhimurium TA1535, in the absence of exogenous metabolic activation ( S9).
In Escherichia coli WP2 uvrA strain equivocal, borderline positive results (revertant colony counts above or just above the genotoxicological threshold for being positive and above the corresponding historical control data range) were obtained at the concentrations of 160 and 50 µg/plate, with addition of S9 (+S9), and the further increased revertant colony numbers followed the clear dose relationship, were above the corresponding historical control data range; however remained below the threshold for being positive at 16 and 5 µg/plate (+S9). Supposedly a genotoxic, but in higher concentrations slightly cytotoxic metabolite of the test item is responsible for these latter changes.
In the performed mutation test, inhibitory effect of the test item on bacterial growth was not observed. All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Conclusions:

Test item induced gene mutations by base-pair substitution in the genome of the strains of Salmonella typhimurium TA100 and by frameshifs in Salmonella typhimurium TA98 and TA1537.
In conclusion, test item has mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1537 carrying base-pair substitution and frameshift mutation in the absence and presence of exogenous metabolic activation, under test conditions used in this study.

Executive summary:

Test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

Bacterial strains, exogenous metabolic activation

Experiments were carried out using S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvrA in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats.

Experimental phases

The study included preliminary solubility trials, a preliminary concentration range finding test (informatory toxicity test applying the plate incorporation method), an initial mutation test (plate incorporation test).

The present test item contains azo-bonds. Therefore, the modified protocol proposed by Prival and Mitchell [10] (e.g.: using of hamster liver S9 Mix that contains 30 % hamster liver extract etc.) was originally planned for the confirmatory mutation test.

However, in this study unequivocal, demonstrative positive results were obtained in the initial mutation test (plate incorporation test); therefore, further investigation, was not necessary for the final conclusion of the study.

Solvent, test item concentrations, rationale for dose selection

Based on the results of the solubility test and the concentration range finding test, the test item was dissolved in dimethyl sulfoxide (DMSO) and the following concentrations were prepared and investigated in the initial mutation testin the absence and presence of exogenous metabolic activation: ±S9: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.

The selection of the concentration range was in accordance with the OECD 471 guideline recommendations, accordingly, the cytotoxicity (absent), the solubility and a possible positive effect of the test item were taken into consideration and the maximum test concentration was in all strains 5000 µg/plate (the recommended maximum test item concentration for soluble non-cytotoxic test items).

At the preparation of the test item suspensions, solutions a correction (multiplier) factor of 1.25 (1/0.8=1.25) based on its purity of 80 % was taken into consideration.

Solubility, precipitation

No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9).

Cytotoxicity results

In the performed experiments inhibitory effect of the test item on bacterial growth was not observed.All of the noticed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding solvent control) remained in the range of the biological variability of the applied test system and the background lawn development was not affected in any case.

Mutagenicity results

The revertant colony numbers of solvent control (dimethyl sulfoxide) plates with and without S9 Mix demonstratedthe characteristic mean number of spontaneous revertantsthat was in line with the corresponding historical control data ranges.The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase)in induced revertant coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains.

In the performed initial mutation test biologically relevant, dose dependently changed revertant colony number increases, positive results were observed in revertant colony numbers of in case of S. typhimurium TA98, TA100 and TA1537 without and with addition of exogenous metabolic activation (±S9) and equivocal borderline positive results (revertant colony counts above or just above the genotoxicological threshold for being positive and above the corresponding historical control data ranges) were obtained in E. coli WP2 uvrA strain in the presence of exogenous metabolic activation (+S9).

The results of the main experiment confirmed well the concentration range finding test results in TA98 and TA100 strains in the absence and the presence of exogenous metabolic activation (±S9). The obtained slight differences (in mutation rates at different concentration levels at these two strains) remained within the biological variability range of the applied test system.

Additionally, in the case of S. typhimurium TA1537 unequivocal, demonstrative positive results were gained in the absence and presence of exogenous metabolic activation (±S9) and equivocal positive results were obtained in E. coli WP2 uvrA, in the presence of exogenous metabolic activation (+S9).

These obtained positive results were demonstrative and enough for the final conclusion of the study.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test iteminduced gene mutations by base-pair substitution and frameshift mutation in the genome of the strains of S. typhimurium TA98, TA100 and TA1537. Equivocal positive results were obtained in E. coli WP2 uvrA.

Conclusion

The reported data of this mutagenicity assay show that under the experimental conditions applied, test item induced gene mutations by base-pair substitution in the genome of the strains of S. typhimurium TA100 and by frameshifts in S. typhimurium TA98 and TA1537.

In conclusion, test item has mutagenic activity on S. typhimurium TA98, TA100 and TA1537 carrying base-pair substitution and frameshift mutation in the absence and presence of exogenous metabolic activation, under the test conditions used in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

supporting study

Study period:

From August 22nd, 1989 to May 9th, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Details on read-across are available in section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

EEC Directive 84/449, L 251, B 10, p. 131-133

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test concentrations with justification for top dose:

EXPERIMENT I
with and without S9 mix:
7 h: 50.0; 100.0; 200.0; 300.0 µg/ml
18 h: 5.0; 25.0; 50.0; 100.0; 200.0; 300.0 µg/ml
28 h: 50.0; 100.0; 200.0; 300.0 µg/ml

Treatment interval was 4 hours. Per experimental point 4 cultures were used in parallel.
According to the criteria mentioned above one (7 h and 28 h) and three concentrations (18 h) were selected to evaluate metaphases for cytogenetic damage.
In the pre-experiment for toxicity the colony forming ability of the V79 cells was at least slightly reduced after treatment with 300.0 g/ml in the absence and presence of S9 mix. In experiment I this concentration was chosen as highest dose level and the respective samples could be scored for cytogenetic damage at each fixation interval.

EXPERIMENT II
With S9
18h: 25.0; 100.0; and 200.0 µg/ml in the presence of S9 mix, samples treated with 300.0 µg/ml could not be evaluated because of severe toxic effects. Therefore, samples treated with 25.0; 100.0; and 200.0 µg/ml were chosen for scoring

Vehicle / solvent:

culture medium (SEROMED, D—1000 Berlin, F.R.G.) without fetal calf serum.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Cultures
The cells have a stable karyotype with a modal chromosome number of 22.
Large stocks of the V79 cell line (supplied by LMP; D-6100 Darmstadt, F.R.G.) are stored in liquid nitrogen in the cell bank of CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch is screened for myco plasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures are propagated at 37 °C in 80 cm2 plastic flasks (GREINER, D-7440 Nürtingen, F.R.G.). Seeding is done with about 5E+05 cells per flask in 15 ml of MEM (minimal essential medium; SEROMED; D-1000 Berlin, F.R.G.) supplemented with 10 % fetal calf serum (FCS; Boehringer Mannheim, D-6800 Mannheim, F.R.G.). The cells are subcultured twice weekly. The cell cultures are incubated at 37 °C and 4.5 % carbon dioxide atmosphere.

PRE-EXPERIMENT FOR TOXICITY
The toxicity of the test article was determined in a pre-experiment in order to establish a concentration dependent plating efficiency relationship. The experimental conditions in this pre experiment were the same as described below for the experiment.


DOSE SELECTION
According to the results from this pre-experiment six concentrations (18 h interval) to be applied in the chromosomal aberration assay were chosen.
The highest dose level used was 10 mM (Collection of notifications related to the pharmaceutical affairs law (IV), Toxicity Test Guideline, Yakugyo Jiho Company, Ltd., Tokyo , Japan, page 43, 1984) unless limited by the solubility of the test article or that producing some indication of cytotoxicity (reduced plating efficiency and/or partial inhibition of mitosis).
If toxic effects were produced the highest dose level should reduce the plating efficiency to approximately 20 - 50 %. In addition, this concentration should suppress if possible mitotic activity (% cells in mitosis) by approximately 50 %, but not so great a reduction that insufficient scorable mitotic cells can be found.


EXPERIMENTAL PERFORMANCE
Seeding of the cultures
Two days old logarithmically growing stock cultures more than 50 % confluent were trypsinised and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution (Trypsin: Difco Laboratories, Detroit, USA).
The cells were seeded into Quadriperm dishes (Heraeus, D-6450 Hanau, F.R.G.) which contained microscopic slides (4 chambers per dish and test group). In each chamber 5 × 10 - 1 × 10 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS.

Treatment
After 48 h (7 h, 28 h preparation interval) and 55 h (18 h pre paration interval) the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 20 µl/ml S9 mix. After 4h this medium was replaced with normal medium after rinsing twice with ‘saline G'.

All incubations were done at 37 °C in a humidified atmosphere with 4.5 % CO2.

Preparation of the cultures
5, 155 and 25.5 h after the start of the treatment colcemid was added (0.2 ug/ml culture medium) to the cultures. 2.0 h (7 h interval) or 2.5 h later, (18 h and 28 h interval) the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KC1) for 20 mm at 37 °C. After incubation in the hypotonic solution the cells are fixed with 3 + 1 absolute methanol + glacial acetic acid. All four slides per group were prepared.
After fixation the cells were stained with giemsa (Merck, D-6100 Darmstadt, F.R.G.).

Evaluation criteria:

Analysis of metaphase cells
Evaluation of the slides were performed using NIKON microscopes with 100× oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per slide were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome number of 22 ±1 °C were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype) is scored.

The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % — 4.00 %.
b) the positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.

A test article is classified as mutagenic if it induces either a significant dose-related increase in the number of structural chromosomal aberrations or a significant and reproducible posi tive response for at least one of the test points.
A test article producing neither a significant dose-related increase in the number of structural chromosomal aberrations nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non-parametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05): evaluated by means of the non-parametric Mann-Whitney test). Evaluation was performed oniy for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The mitotic index was clearly suppressed after treatment with 300.0 µg/ml (without S9 mix) and with 50.0 µg/ml and higher concentrations in the presence of S9 mix (exp. I and II).

In the pre-experiment on toxicity (colony forming ability) after treatment with 10.0 µg/ml and higher (without S9 mix) and 30.0 µg/ml and higher (with S9 mix) the colony forming ability was distinctly reduced as compared to the solvent controls.
In the main experiments, at each fixation interval, except at interval 28 h without S9 mix, the mitotic index was reduced at least after treatment with the highest dose levels in the absence and presence of S9 mix, indicating that the substance had cytotoxic properties.
In the absence of S9 mix the test article did not increase the frequency of cells with aberrations at any fixation interval. The aberration rates of the cells after treatment with the test article (1.25 % - 3.25 %) were in the range of the control valu es: 1.25 % - 3.75 %.

However, in the presence of S9 mix, at fixation interval 18 h a statistically significant increase in the aberration rate (9.25 %) was found after treatment with 100.0 µg/ml. To clarify this none dose-dependent result a second experiment was performed.
In this experiment samples only treated with 200.0 µg/ml of the test article could be evaluated as top dose level because of severe toxic effects with 300.0 µg/ml. In the samples treated with 200.0 µg/ml again a slight but statistically significant
increase in the aberration rate (5.75 %) as compared to the solvent control (1.75 %) was observed. As the aberration rate was beyond the range of the control data of this study (1.25 % -3.75 %) and of our historical control data (0.00 % - 4.00 %) this increase is regarded as biologically relevant.

Additional indications for effects of the test article on chromatin arrangement arise from occurrence of abnormal interphase nuclei. There were many multinucleate cells carrying a large number of nuclei of various sizes within a single cell. Some cells in mitosis were observed of which the chromosomes appeared clustered in groups of different sizes. Also, a distinct number of interphase nuclei were observed with an abnormal condensed chromatin pattern (in clusters).

These observations, together with the distinct decreased mitotic index and the slightly increased aberration frequency can lead to the assumption that the test article probably do not only interact directly with the chromatin but also affect the cell cycle and disarranged the development of chromatin condensation.

No relevant deviation from the control data was found after treatment with the test article.
EMS (0.72 mg/ml) and CPA (1.40 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Positive
The test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.
Therefore, it is considered to be mutagenic in this chromosomal aberration test.

Executive summary:

Materials and methods

The test article was assessed for its potential induce to structural chromosome aberrations in V79 cells of the Chinese hamster in vitro, according to OECD guideline 473.

Preparation of chromosomes was done 7 h (high dose), 18 h (low, medium and high dose), and 28 h (high dose) after start of treatment

with the test article. The treatment interval was 4 h.

In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 metaphases were scored for structural chromosomal aberrations.

The following dose levels were evaluated:

Experiment I, with and without S9 mix:

7 h: 300 µg/l

18 h: 25.0; 100.0; 300.0 µg/ml

28 h: 300.0 µg/ml

Experiment II, with S9 mix:

18 h: 25.0; 100.0; 200.0 µg/ml

The concentration range of the test article applied had been determined in a pre-experiment using the plating efficiency assay as indicator for toxicity response. Treatment with 200.0 and 300.0 µg/ml reduced clearly the plating efficiency of the V79 cells (derived from the plotted dose re sponse curve).

Also the mitotic index was at least slightly reduced after treatment with 300.0 µg/ml (without S9 mix) and with 50.0 µg/ml and higher in the presence of S9 mix (exp. I and II).

There was a slight but statistically significant increase in cells with structural aberrations after treatment with 100.0 (exp. I) and 200.0 µg/ml (exp. II) at fixation interval 18 h with metabolic activation by S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article induced structural chromosome aberrations as determined by the chromosomal aberration test in the V79 Chinese hamster cell line.

Therefore, it is considered to be mutagenic in this chromosomal aberration test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Mammalian erythrocytes micronucleus assay (OECD 474): negative

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Study period:

From August 29th to November 7th, 1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Details on read-across are available in section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

EEC Directive 84/449, L 251, B 12, p. 137 - 139

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Strain: NNRI
Source: BRL Tierfarm Füllinsdorf CH- 4414 Füllinsdorf/Basel, Switzerland
Number of animals: 84 (42 males/42 females)
Initial age at start of acclimatization: minimum 10 weeks
Acclimatization: minimum 5 days
Initial body weight at start of treatment: approximately 30 g
According to the suppliers assurance the animals were in healthy condition.
The animals were under quarantine in the animal house of CCR for one week after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

HUSBANDRY
Housing: single
Cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-7830 Einmendingen)
Bedding: granulated soft wood bedding (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Feed: pelleted standard diet (ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
Water: tap water, ad libitum (Gemeindewerke, D61O1 Rossdorf, F .R.G.)
Enviromnent: temperature 21 ± 3 °C
Relative humidity 30-70 %
Artificial light 6.00 a.m.- 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

The vehicle of the test article was used as negative control.
Carlioxymethylcellulose
Source: SERVA, D-6900 Heidelberg
Batch: 17681
Somministration: orally, singly
Volume: 10 ml/kg b.w.

Details on exposure:

DOSE SELECTION
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibility. The volume to be administered should be compatible with physiological space available.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on sur vival within 72 hours.

Duration of treatment / exposure:

24, 48 and 72 hours

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

pre-experiment I

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Remarks:

pre-experiment II

Dose / conc.:

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

Remarks:

pre-experiment II

No. of animals per sex per dose:

6 males, 6 females

Positive control(s):

CPA; Cyclophosphamide
Source: SERVA, D-6900 Heidelberg, F.R.G.
Batch: 17681
Dissolved in: physiological saline
Somministration: 40 mg/kg b.w., orally, singly
Volume: 10 ml/kg b.w.
Solution prepared on day of administration.
The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolised per day in aqueous solution.

Tissues and cell types examined:

To describe a cytotoxic effect the ratio between polychromatic and normochrcmatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Details of tissue and slide preparation:

Evaluation of the slides was performed using NIKON microscopes with 100× oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polycliromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05)
was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

pre-experiment I (4 males/4 females 5000 mg/kg bw)
All surviving animals expressed toxic reactions: reduction of spontaneous activity, eyelid closure and apathy. One male and one female died within the first hour after treatment.
pre-experiment II (2 males/2 females 1500 mg/lg bw)
All treated animals expressed toxic reactions: reduction of spontaneous activity. One female expressed abdominal position.
Additionally, one male and one female expressed eyelid closure and apathy.
2000 mg/kg bw
All treated animals died within 6 hours after treatment.

After treatment with 1500 mg/kg bw of the substance one male and one female died at preparation interval 24 hours, one female died at preparation interval 48 hours, and one female died at prepa ration interval 72 hours.
The mean number of normochromatic erythrocytes was not increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating no cytotoxic properties.

In comparison to the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with were in the same range as compared to the negative control groups.

Conclusions:

During the study described and under the experimental conditions reported, test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
Therefore, the substance is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of mouse. Test substance was suspended in Carboxymethylcellulose (1 %). This suspending agent was used as negative control. The volume administered orally was 10 ml/kg bw.

24 h, 48 h and 72 h after a single application of test substance, bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with test substance, the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.

The following dose level of the test article was investigated: 24 h, 48 h, and 72 h preparation interval: 1500 mg/kg bw.

In pre-experiments this dose level was estimated to be the maximum tolerated dose. The animals expressed toxic reactions.

After treatment with test substance, the ratio between PCEs and NCEs was not affected as compared to the corresponding negative

controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls, there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article.

An appropriate reference mutagen was used as positive control, which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, test substance

did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The assessment on the potential genetic toxicity of Acid Brown 303 relied on both data on the substance itself and on a structural analogue, i.e. Similar Substance 01. Details on the read across are reported in section 13.

Acid Brown 303 was tested according to OECD guideline 471 in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and E. coli WP2 uvrA in presence and absence of metabolic activation. Controls were runb in parallel. Acid Brown 303 was found to be mutagenic in this assay.

Due to the positive response in the bacterial mutation assay, an available chromosomal aberration assay run on Similar Substance 01 was taken into consideration. The potential of Similar Substance 01 to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro was assessed according to OECD guideline 473.

The substance induced structural chromosome aberrations, thus it is considered to be positive in this assay.

To complete the assessment, an available in vivo study on Similar Substance 01 was taken into account. This study was run to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, according to OECD guideline 474.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE. The following dose level of the test article was investigated:

24 h, 48 h, and 72 h preparation interval: 1500 mg/kg bw.

In pre-experiments the animals expressed toxic reactions.

After treatment with the test article the ratio between PCEs and NCEs was not affected as compared to the negative controls.

Under experimental conditions, the substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

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

The available studies fulfil the assessment strategy for genotoxic potential as described in the ECHA Guidance R.7a.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), classification for genetic toxicity is as follows:

Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans

The overall evaluation of the potential of Acid Brown 303 to induce genetic toxicity was based on: positive responses in a bacterial reverse mutation assay and in vitro chromosomal aberration assay; negative response in an in vivo micronucleus assay.

According to ECHA Guidance R.7a, positive in vitro outcomes need to be further investigated in representative in vivo assays. As the in vivo micronucleus outcome was negative, Acid Brown 303 may be considered as devoid of a genotoxic potential and it thus is not classified within the CLP Regulation (EC 1272/2008).