Methyl 4-cyano-5-[[5-cyano-2,6-bis[(3-methoxypropyl)amino]-4-methyl-3-pyridyl]azo]-3-methyl-2-thenoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In all available Ames tests Macrolex Rot B was negative S. typhimurium strains TA 100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with and without metabolic activation.

Macrolex Rot B did not induce gene mutations in an HPRT assay according to OECD guideline 476 in V79 cells of the Chinese hamster. Macrolex Rot B was negative (non-mutagenic) in this HPRT assay.

In an OECD guideline 487 (In vitro Mammalian Cell Micronucleus Test) study, Macrolex Rot B did not induce micronuclei as determined by the in-vitro micronucleus test in Chinese hamster V79 cells. Therefore, Macrolex Rot B is considered to be non-mutagenic in this in-vitro micronucleus test, when tested up to precipitating concentrations.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The ability of MACROLEX Rot B to induce mutations was investigated using Salmonella typhimurium strains TA 100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the absence and presence of a metabolic activation System (S9 mix).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Chemical name. Methyl 4-cyano-5 [[5-cyano-2,6-bis[(3-methoxy propyl)amino]-4-methyl-3-pyridyl azo]-3-methyl-2-thenoate
Other name: MACROLEX Rot B
CAS Number: 72968-71-9
Purity: >99% (w/w)

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:

S-9 mix

Test concentrations with justification for top dose:

Negative control, 0.305, 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-(2-FuryI)-3-(5-nitro-2 furyl)acrylamide, Sodium azide, 2-Aminoanthracene

Details on test system and experimental conditions:

This study was performed by the pre-incubation method without and with S9 mix. Triplicate plates were used for the negative control group, and duplicate plates were used for the positive control and the test substance treatment groups.

Procedures
After 0.1 mL of the test substance solution, vehicle or the positive control substance solution, 0.5 mL of 0.1 M sodium phosphate bufier (pH 7.4) or S9 mix and 0.1 mL of the bacterial culture were added to a test tube, the mixture as shaken at 37±0.5°C for 20 minutes. Two milliliters of the soft agar were then added to each tube and the mixture was poured onto a minimal glucose agar plate. The number of revertant colonies was counted after incubation at 37±0.5°C for 48 hours.

Confirmation of Sterility
The highest concentration of the test substance solution (0.1 mL) and S9 mix (0.5 mL) were individually mixed with 2 mL of the soft agar and were poured onto the minimal glucose agar plate in order to examine bacterial contamination.
Bacterial contamination was judged with those plates after incubation at 37±0.5°C for 48 hours.

Negative Control and Positive Controls
The vehicle was employed as a negative control. As positive controls 2-(2-FuryI)-3-(5-nitro-2 furyl)acrylamide, Sodium azide, 2-Aminoanthracene were used.

Evaluation criteria:

The test substance was judged to be positive when the number of revertant colonies increased to twice or more than that in the negative control and when the responses were dose-related and/or reproducible. The other cases were judged to be negative. No statistical methods were used.

Key result

Species / strain:

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

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:

The mutagenicity of the test substance was judged to be negative because the number of the revertant colonies in the test substance treatment groups in all tester strains was less than twice that in the negative control without and with S9 mix.
The numbers of the revertant colonies in the positive controls were above twice that in the negative controls, The test results showed that the numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data at the testing facility. It was also confirmed that no bacterial contamination was observed, which indicates the test results to be valid.

Conclusions:

It was concluded that MACROLEX Rot B did not induce mutations under the present test conditions.

Executive summary:

The ability of MACROLEX Rot B to induce mutations was investigated using Salmonella typhimurium strains TA 100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the absence and presence of a metabolic activation System (S9 mix).

As a result, the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all testet strains with and without S9 mix.

Consequently, it was concluded that MACROLEX Rot B had no ability to induce mutations under the present test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Principles of method if other than guideline:

The substance Thermoplast Rot 454 was tested for its mutagenic potential based on the ability to induce point mutations in selected Ioci of several bacterial strains (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA), in a reverse mutation assay (Ames standard plate test and Prival preincubation test) with and without metabolic activation.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name of test substance: Thermoplast Rot 454
CAS No.: 72968-71 -9
Purity: 99.7%%

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:

S-9 mix

Test concentrations with justification for top dose:

DOSE RANGE:
20 µg - 5 000 µg/plate (SPT)
20 µg - 5 000 µg/plate (PIT)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, congo red, benzidine, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylenediamine, 9-aminoacridine, 4-nitroquinoline-N-oxide

Details on test system and experimental conditions:

EXPERIMENTAL PROCEDURE
Choice of the vehicle
Due to the insolubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Mutagenicity tests
Ames standard plate test
Salmonella typhimurjum
The experimental procedure of the standard plate test (plate incorporation method) is based on the method ofAmes et al. Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml purified water
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli
The experimental procedure is based on the method of Ames et al. Test tubes containing 2-ml portions of soft agar (overlay agar), which consists of 100 ml agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino
acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 ml test solution or vehicle
0.1 ml fresh bacterial culture
0.5 ml S-9 mix (in tests with metabolic activation) or 0.5 ml phosphate buffer (in tests without metabolic activation)

After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
Composition of the minimal agar:
Ihe composition of the minimal agar (SAI selective agar) is based on the description of Green, M.H.L. and Muriel, W.J.,with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 ml solution B (agar)
100 ml solution A (sahne solution)
8 ml solution C (glucose solution)
10 ml solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

Prival preincubation test
The experimental procedure is based on the method of Yahagi et al. and Matsushima et al. and has been modified further to include reductive conditions by Prival et al.
0.1 ml test solution or vehicle, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 30°C for 30 minutes using a shaker. Subsequently, 2 ml of soft agar which consists of 100 ml agar (0.8% agar + 0.6% NaCI) and 10 ml amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin or 0.5 mM tryptophan) is added. After mixing, the samples are poured onto the Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml purified water
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
5 g D-glucose, monohydrate.
After incubation at 37°0 for 48 - 72 hours in the dark, the bacterial colonies (his+ and trp+ revertants) are counted.

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either withaut S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

Key result

Species / strain:

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

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:

According to the results of the present study, the test substance Thermoplast Rot 454 is negative (not mutagenic) in the Ames standard plate test and in the Prival preincubation test under the experimental conditions chosen here.

Conclusions:

According to the results of the present study, the test substance Thermoplast Rot 454 is negative (not mutagenic) in the Ames standard plate test and in the Prival preincubation test under the experimental conditions chosen here.

Executive summary:

The substance Thermoplast Rot 454 was tested for its mutagenic potential based on the ability to induce point mutations in selected Ioci of several bacterial strains, i.e. Salmonella typhimurlum and Escherichia coIi, in a reverse mutation assay (Ames standard plate test and Prival preincubation test).

- STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

- DOSE RANGE: 20 µg - 5 000 µg/plate (SPT); 20 µg - 5 000 µg/plate (PIT)

- TEST CONDITIONS: Ames standard plate test (SPT) with and without metabolic activation (Aroclor-induced rat liver S-9 mix) and Prival preincubation test (Ph) with and without metabolic

activation (uninduced hamster liver S-9 mix).

- SOLUBILITY: Precipitation of the test substance was found from about 100 µg/plate onward.

- TOXICITY: No bacteriotoxic effect was observed.

- MUTAGENICITY:

An increase in the number of his+ or trp+ revertants was not observed either in the Ames standard plate test or in the Prival preincubation test without S-9 mix or after the addition of a metabolizing system.

- CONCLUSION:

According to the results of the present study, the test substance Thermoplast Rot 454 is not mutagenic in the Ames standard plate test and in the Prival preincubation test under the experimental conditions chosen here.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 18 May 2016 Experimental completion date 07 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian forward mutation assay

Specific details on test material used for the study:

Appearance: Red powder
Storage Conditions: At room temperature
Expiry Date: 13 November 2020
Stability in Solvent: 0.2 and 250 mg/mL stable for 4 h and 24 hours in DMSO

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22.

Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 75 cm2 plastic flasks. 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%). The cells were sub-cultured once or twice weekly.
All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.

For seeding of the cell cultures the complete culture medium was 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. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 was used as metabolic activation system.

Test concentrations with justification for top dose:

Pre-experiment toxicity test
With and without S9-mix: 10.6, 21.3, 42.5, 85.0, 170.0, 340.0, 680.0, 1360.0 µg/ml
The maximum concentration in the pre-test was 1360 µg/mL based on the solubility properties of the test item in and aqueous medium.

No relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the beginning and at the end of treatment (4 hours) prior to removal to the test item. Precipitation was noted at 170 µg/mL and above after 4 hours treatment with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.

Main experiment:
With and without S9-mix: 10.6, 21.3, 42.5, 85.0, 170.0, and 340 µg/ml
The dose range of the main experiment was set according to data generated in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.
To overcome problems with possible deviations in toxicity or solubility the main experiment was started with more than four concentrations.

Vehicle / solvent:

The vehicle was DMSO. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 300 μg/mL = 2.4 mM

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

Final concentration: 2.3 μg/mL = 8.9 μM

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Experimental Design and Study Conduct
Pre-Test on Toxicity
A pre-test was performed in order to determine the toxicity of the test item. The maximum concentration in the pre-test was 1360 µg/mL based on the solubility properties of the test item in and aqueous medium.
In addition the pH-value and the osmolarity were measured. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment.
In the pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is evident as a reduction of the cloning efficiency (CE).

Experimental Performance
The experiment was performed with a treatment time of 4 hours with and without metabolic activation. Two parallel cultures were used throughout the assay.

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.

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. Concurrent solvent and positive controls were treated in parallel. 4 hours after treatment, this medium was replaced with complete medium following two washing steps with "saline G".
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 as described below.
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.

Evaluation criteria:

HPRT catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Therefore, cells deficient in HPRT due to a forward mutation are resistant to 6-TG. These cells are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.

A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression analysis (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. However, both, biological and statistical significance was considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Macrolex Rot B was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

In the main experiment precipitation at the end of treatment was observed at 85.0 µg/mL and above with and without metabolic activation.
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was slightly exceeded at 85.0 µg/mL in culture I with metabolic activation but the corresponding t-test was not significant.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. A t-test performed at 85.0 µg/mL over both parallel cultures with metabolic activation was not significant.
In the main experiment with and without S9 mix the range of the solvent controls was from 9.5 up to 26.7 mutants per 10^6 cells; the range of the groups treated with the test item was from 10.8 up to 29.8 mutants per 10^6 cells.
EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

In conclusion it can be stated that under the experimental conditions reported Macrolex Rot B did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, Macrolex Rot B is considered to be non-mutagenic in this HPRT assay.

Executive summary:

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

The treatment period was 4 hours with and without metabolic activation.

The maximum concentration of the pre-test on toxicity (1360 µg/mL) was limited by the solubility of the test item. The dose range of the main experiment was limited by precipitation of the test item in aqueous media.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

Conclusion

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

Therefore, Macrolex Rot B is considered to be non-mutagenic in this HPRT assay.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 27 September 2016 Experimental completion date: 07 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Expiry Date: 13 November 2020
Storage Conditions: At room temperature
Appearance: Red powder
No correction for purity was made.

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate (doubling time of V79 cells in stock cultures: approximately 13 hours, determined on December 17, 2010) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 1.

Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH. This allows the repeated use of the same cell culture batch in experiments. Before freezing each batch is screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells.

Culture conditions
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 10^5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts, glutamine and Hepes (25 mM). Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 µg/mL) and 10 % (v/v) fetal bovine serum (FBS). The cells were sub-cultured twice a week.

Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment was stopped and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.25 % (w/v) in Ca-Mg-free salt solution. Per culture approximately 5.0 – 6.0 x 10^5 cells were seeded into 25 cm2 plastic flasks.

All incubations were done at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Cytokinesis block (if used):

cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment:
I (4 hour exposure period, with and without S9-mix) - 1.7, 3.4, 6.8, 13.6, 27.2, 54.4, 109, 218, 544 and 1360 µg/ml
II (24 hour exposure without S9-mix) -6.2, 10.8, 18.9, 33.1, 58.0, 102, 178, 331, 544 and 1360 µg/ml

Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test.

Vehicle / solvent:

Stock formulations of the test item and serial dilutions were made in DMSO. The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The final concentration of DMSO in the culture medium was 1.0 %.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.1 µg/ml

Positive control substance:

mitomycin C

Remarks:

Pulse treatment, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

7.0 µg/ml

Positive control substance:

other: Griseofulvin

Remarks:

Continuous treatment, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

4.0 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

Pulse treatment, with metabolic activation

Details on test system and experimental conditions:

The induction of cytogenetic damage in V79 cells (Chinese hamster cell line) was assessed in two independent experiments with one preparation interval (24 hours).

Pre-experiment
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterized by the percentages of reduction in the Cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture. The experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
The pre-test was performed with 10 concentrations of the test item separated by no more than a factor of √10 and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 24 hrs after start of the exposure.

Cytogenetic Experiment
Pulse exposure
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL culture medium was added. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose ∙ H2O, 192 mg/L Na2HPO4 ∙ 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 20 hours.

Continuous exposure (without S9 mix)
The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item. At the same time Cytochalasin B was added to the cell culture (1.5 µg/mL). The medium was not changed until preparation of the cells.

Preparation of cells
Cells were detached by trypsin-EDTA-solution for approx. 5 minutes, followed by stopping the enzymatic treatment by adding complete culture medium including 10 % (v/v) FBS. The cultures were harvest and spun down by gentle centrifugation for 7 min. The supernatant was discarded and the cells were resuspended in saline G and spun down once again by centrifugation. Then the cells were resuspended in KCL solution (0.4 %) and incubated at 37°C for 10 minutes. Ice-cold fixative mixture of methanol and glacial acetic acid (19+1 parts, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the supernatant after centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping a small amount of the cell suspension in fresh fixative on clean, wet microscope slides and allowed to dry. The slides were stained with Giemsa, mounted after drying and covered with a cover slip. All slides were labeled with a computer-generated random code to prevent scorer bias.

Evaluation criteria:

A test item is considered to be clearly negative if, in all of the experimental conditions examined:
- None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- There is no concentration-related increase
- The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data

There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.

However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test item Macrolex Rot B, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamster V79 cells in vitro in the absence and presence of metabolic activation by S9 mix.
Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 24 hours without S9 mix. The cells were prepared 24 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 1000 cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined.
The highest treatment concentration in this study, 1360 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.
Precipitation of the test item in the culture medium was observed in Experiment I at 109 µg/mL and above in the absence and presence of S9 mix and in Experiment II at 102 µg/mL and above in the absence of S9 mix at the end of treatment.
No relevant influence on osmolarity or pH was observed. The osmolarity is generally high compared to the physiological level of approximately 300 mOsm. This effect however, is based on a final concentration of 1% DMSO in medium. As the osmolarity is measured by freezing point reduction, 1% of DMSO has a substantial impact on the determination of osmolarity.
In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.
In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item.
In Experiment I in the presence of S9 mix after treatment with 54.4 µg/mL, the value of 2.30 % micronucleate cells exceeded the range of the historical solvent control data (0.0 – 2.10 % micronucleate cells), but was not statistical significant. No significant increase was observed at the highest concentration (109 µg/mL) which showed precipitation. Overall, the finding is regarded as biologically irrelevant.
In both experiments, either Griseofulvin (7.0 µg/mL), MMC (0.1 µg/mL) or CPA (4.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

Concentrations applied

Exp.

Prep. interval

Exposure period

Concentrations in µg/mL

Without S9 mix

I

24 hrs

4 hrs

1.7

3.4

6.8

13.6

27.2

54.4

109^P

218^P

544^P

1360^P

II

24 hrs

24 hrs

6.2

10.8

18.9

33.1

58.0

102^P

178^P

311^P

544^P

1360^P

With S9 mix

I

24 hrs

4 hrs

1.7

3.4

6.8

13.6

27.2

54.4

109^P

218^P

544^P

1360^P

        Evaluated experimental points are shown in bold characters

^P       Precipitation was observed microscopically at the end of treatment

Summary of results

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	24 hrs	Solvent control^1	1.93		1.60
		Positive control^2	1.51	44.9	16.30^S
		27.2	1.98	n.c.	1.65
		54.4	1.96	n.c.	1.65
		109^P	1.95	n.c.	2.15
Exposure period 24 hrs without S9 mix
II	24 hrs	Solvent control^1	1.87		1.45
		Positive control^3	1.86	1.4	7.95^S
		33.1	1.93	n.c.	0.85
		58.0	1.90	n.c.	1.00
		102^P	1.85	2.6	0.85
Exposure period 4 hrs with S9 mix
I	24 hrs	Solvent control^1	1.94		1.55
		Positive control^4	1.83	11.6	4.55^S
		27.2	1.94	0.1	1.60
		54.4	1.93	1.1	2.30
		109^P	1.93	1.7	1.40

*      For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

^P       Precipitation occurred at the end of treatment

^S       The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c.   Not calculated as the CBPI is equal or higher than the solvent control value

¹       DMSO                1.0 % (v/v)
²       MMC                 0.1 µg/mL

³       Griseofulvin       7.0 µg/mL

⁴       CPA                   4.0 µg/mL

 

 

 

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in Chinese hamster V79 cells.
Therefore, Macrolex Rot B is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Executive summary:

The test item Macrolex Rot B, suspended in DMSO, was assessed for its potential to induce micronuclei in Chinese hamsterV79cellsin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Exposure period	4 hrs	24 hrs	4 hrs
Recovery	20 hrs	¾	20 hrs
Preparation interval	24 hrs	24 hrs	24 hrs

In each experimental group two parallel cultures were analyzed. Per culture 1000 cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1360 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 487. The chosen treatment concentrations are reported inTable 1and the results are summarized inTable 2.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In the absence and presence of S9 mix, no relevant increases in the number of micronucleate cells were observed after treatment with the test item.

In Experiment I in the presence of S9 mix after treatment with 54.4 µg/mL, the value of 2.30 % micronucleate cells exceeded the range of the historical solvent control data (0.0 – 2.10 % micronucleate cells), but was not statistical significant. No significant increase was observed at the highest concentration (109 µg/mL), which showed precipitation. Overall, the finding is regarded as biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

Conclusion

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by thein vitro micronucleus test in Chinese hamster V79 cells.

Therefore, Macrolex Rot B is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to precipitating concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Macrolex Rot B was non-mutagenic (negative) in all in vitro tests. In all available Ames tests, in an HPRT assay according to OECD guideline 476 in V79 cells and an in vitro Mammalian Cell Micronucleus Test according to OECD guideline 487 Macrolex Rot B was negative. According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is therefore not justified.