Bisbenzimidazo[2,1-b:2',1'-i]benzo[lmn][3,8]phenanthroline-8,17-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Orange 43 did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537; E. coli WP2 uvrA) with and without metabolic activation (rat liver S9) at up to 5000 µg/plate. Therefore Pigment Orange 43 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

Pigment Orange 43 was not genotoxic in the mammalian cell gene mutation test (HPRT) in Chinese Hamster ovary cells (CHO-K1) when tested with and without rat liver S9 metabolic activation at concentrations up to 50 µg/mL.

 

Pigment Orange 43 was not clastogenic in the mammalian cell chromosome aberration test in Chinese Hamster ovary cell (CHO-Kl) when tested with and without rat liver S9 metabolic activation at concentrations up to 12 µg/mL.

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)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- for S. typhimurium strains TA 1537, TA 1535, TA 100, TA 98
trp- for E. coli WP2 uvr A

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:

phenobarbital/ß-naphthoflavone-induced rat liver S9-mix

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Experiment II (pre-incubation): 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity > 99 % (Merck Darmstadt, Germany)

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide, 4-nitro-o-phenylenediamine, methylmethanesulfonate / With metabolic activation: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a preincubation assay.

DURATION
- Preincubation period: 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: yes

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not mandatory according to OECD guideline 471

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with metabolic activation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Slight toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with metabolic activation at 1000 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all experiments.

In experiment I slight toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5, occurred in the strains TA 98 and TA 1537 in the test groups with metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Due to a new evaluation unit, new historical control data are evaluated since July 2004 representing 80 experiments. This deviation to the study plan had no detrimental impact on the outcome of the study.

The historical control range was exceeded in strains TA 1535 (experiment I with metabolic activation). This effect indicates the sensitivity of the strains rather than compromising the assay.

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

Conclusions:

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

Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Executive summary:

This study was performed to investigate the potential of Pigment Orange 43 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed according to OECD TG 471 with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. Slight toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5, occurred in the strains TA 98 and TA 1537 in the test groups with metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Pigment Orange 43 at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese Hamster (Cricetulus grise us) ovary cell line CHO-Kl, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.

Metabolic activation:

with and without

Metabolic activation system:

Aroc1or 1254 induced rat liver S9 homogenate

Test concentrations with justification for top dose:

Based on the observations of the preliminary cytotoxicity test, following concentrations of Hostaperm-Orange GR were selected for testing in the initial chromosomal aberration assay.

Experiments No.1 and No.2: 3, 6, and 12 µg/mL

Experiment No.3: 2, 4 , and 8 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.

Batch No. 3 of this CHO-K1 cell line was tested for the absence of mycoplasma contamination at Mycoplasma Laboratory, Statens Serum Institut, Artillerivej 5, Copenhagen S, Denmark and certified free of mycoplasma contamination on 11 August 2014.

Cells were grown in T-75 cm2 flasks at 37 ± 1 °C in a carbon dioxide incubator (5 ± 0.2 % CO2 in air).

Evaluation criteria:

When all the validity criteria are fulfilled:
 
1.        A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control
- The increase is dose-dependent when evaluated with an appropriate trend test
- Any of the results are outside the distribution of the historical vehicle control data
 
2.        A test chemical is considered to be clearly negative if, in all experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in aberrant metaphases compared with the concurrent vehicle control
- There is no concentration-related increase when evaluated with an appropriate trend test
- All results are inside the distribution of the historical vehicle control data
 
3.        The results are considered equivocal if they do not meet the criteria specified for a positive or negative response. Additional experimental work may be required by the Study Director to clarify such results and the activity, if any, will be documented in a study plan amendment.

Statistics:

Statistical analysis of the experimental data was carried out using validated SYSTAT Statistical package ver.12.0. Data were analyzed for proportions of aberrant metaphases in each sample excluding gaps as aberrations, numerical aberration (Poly) were analyzed. Pooled data from each test concentration and the positive control were compared with the vehicle control using Fisher exact test. All analysis and comparisons were evaluated at 5 % (p < 0.05) level.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

       

  Results

a)  Sterility Check

 

The S-9 homogenate was found to be sterile.

 

b)  Metabolic Activation

 

The S-9 homogenate was characterized for its ability to metabolize the promutagens 2-aminoanthracene and benzo(a)pyrene to mutagens using Salmonella typhimurium TA100 strain and the S-9 homogenate was found to be active.

 

c)  Protein Content

 

The protein content of the S-9 homogenate was 30 mg/mL and was found to be satisfactory.

                      

The test item formeda free flowing suspension in DMSO at 50 mg/mL.

 

1. Solubility Test and Justification for the Selection of Vehicle

DMSO is one of the organic vehicles compatible with this test system. Hence, based on the results of solubility test, DMSO was selected as the vehicle of choice to prepare the stock and dilutions of the test item.

2. Cytotoxicity Test and Justification for the Selection of Test Doses

At the end of 3-hour exposure, Hostaperm-Orange GR did not precipitate in the test medium at any of the tested concentrations.

 

At the end of 3-hour exposure, the pH of the test medium in the presence of metabolic activation ranged from 6.99 to 7.10 with 7.00 in the DMSO control while in the absence of metabolic activation it was between 7.05 and 7.17 with 7.15 in the DMSO control.

 

At the end of 3-hour exposure, osmolality of the test medium at the highest treatment condition (500mg/mL) was 0.412 and 0.386 OSMOL/kg, in the presence and absence of metabolic activation, respectively, whereas the corresponding osmolality in the DMSO control was 0.412 and 0.401 OSMOL/kg, respectively.

 

Hostaperm-Orange GR was toxic to the CHO-K1 cells at and above 20 µg/mL where the cell counts were negligible. At 10 µg/mL, the cell growth inhibition as RICC was 49 and 44% in the presence and absence of metabolic activation with 3-hour exposure and 55% in the absence of metabolic activation with 21-hour exposure.

 

Based on these observations, in the chromosomal aberration assay, a maximum of 12 µg/mL was tested in Experiments 1 and 2, and a maximum of 8 µg/mL was tested in Experiment 3.

3. Chromosomal Aberration Assay

Experiment 1

 

At the highest concentration tested (12 µg/mL), the reduction in the cell growth was 51 % compared to the vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

The positive control, cyclophosphamide monohydrate caused a statistically significant increase in the aberrant metaphases excluding gaps.

 

 Experiment 2

 

At the highest concentration tested (12 µg/mL), the reduction in the cell growth was 42 % compared to the vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the concentrations tested.

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

Experiment 3

 

At the highest concentration tested (8 µg/mL), the reduction in the cell growth was 56 % compared to the respective vehicle control.

 

The incidence of aberrations in the vehicle control was within the range of the in-house historical control data.

 

The incidence of aberrant metaphases excluding gaps was statistically comparable to the vehicle control value at all the three test concentrations. 

 

No incidences of polyploidy, endoreduplication and pulverization observed.

 

Ethyl methanesulphonate caused a statistically significant increase in the aberrant metaphases excluding gaps.

DISCUSSION

The assay conditions fulfilled all the specifications as per the OECD 473 guideline.

 

There was no evidence of an increase in either structural or numerical aberrations in cultures treated with Hostaperm-Orange GR either in the presence or in the absence of metabolic activation.

 

The results of this study suggest that the test item, Hostaperm-Orange GR does not have the potential to cause chromosome damage in the presence or absence of metabolic activation.

 

In each of these experiments, the respective positive controls produced a statistically significant increase in aberrant metaphases, demonstrating that the system was able to detect the effect of known mutagens.

 

Conclusions:

It was concluded that the test item was not clastogenic in CHO cells at the tested concentrations and under the conditions of testing employed.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Executive summary:

The clastogenic potential of the test item to induce chromosomal aberrations in mammalian cells was evaluated according to OECD TG 473 using cultured Chinese Hamster Ovary (CHO) cells in the presence and absence of an exogenous metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

The study consisted of a preliminary cytotoxicity test and a chromosomal aberration assay. Chromosomal aberration assay consisted of three independent experiments: Experiments 1 and 2 in the presence and absence of metabolic activation system with 3-hour exposure, respectively, and Experiment 3 in the absence of metabolic activation system with 21-hour exposure.

 

The test item formed a free flowing suspension in DMSO at 50 mg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the chromosomal aberration assay, the test item was toxic to the CHO-K1 cells at and above 20 µg/mL where the cell counts were negligible. At 10 µg/mL, the cell growth inhibition as RICC was 49 and 44% in the presence and absence of metabolic activation with 3-hour exposure and 55% in the absence of metabolic activation with 21-hour exposure. The test item did not precipitate in the test medium and did not cause any appreciable change in the pH and Osmolality of test medium. Based on these observations, in the chromosomal aberration assay, a maximum of 12 µg/mL was tested in Experiments 1 and 2, and a maximum of 8 µg/mL was tested in Experiment 3.

 

In Experiments 1 and 2 of the chromosomal aberration assay, CHO-K1 cells were exposed to the test item in duplicate for 3 hours at concentrations of 3, 6 and 12 µg/mL in the presence and absence of metabolic activation, respectively. In Experiment 3, CHO-K1 cells were exposed to the test item in duplicate for 21 hours at concentrations of 2, 4 and 8 µg/mL in the absence of metabolic activation. Concurrent vehicle (DMSO) and positive controls (cyclophosphamide monohydrate in the presence of metabolic activation and ethyl methanesulfonate in the absence of metabolic activation) were also tested in duplicate. In each case, the cells in C-metaphase were harvested at 21 hours after the start of the treatment and slides were prepared for chromosomal analysis.

 

At the highest concentration tested, the reduction in cell growth as RICC was 51, 42 and 56 % in Experiments 1, 2 and 3, respectively, compared to the vehicle control.

 

A total of 300 metaphases each from the DMSO control, each treatment level and the positive control were evaluated for chromosomal aberrations.

 

There was no evidence of induction of chromosome aberrations, excluding gaps, either in the presence or in the absence of metabolic activation. There was also no incidence of polyploid cells indicating that the test item did not induce numerical chromosomal aberrations. In each of these experiments, under identical conditions, the respective positive control substances produced a large and statistically significant increase in aberrant metaphases.

 

The study indicated that the test item was not clastogenic at the concentrations tested and under the conditions of testing.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

HPRT

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test concentrations with justification for top dose:

Based on the results of the preliminary cytotoxicity test, the following test concentrations were selected for testing in the gene mutation test:

Experiments No.1 and No.2 (Presence and Absence of Metabolic Activation, respectively): 2, 6, 17, and 50 µg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Details on test system and experimental conditions:

Chinese Hamster (Cricetulus griseus) ovary cell line CHO-K1, (ATCC CCL-61, Lot 4765275) with a modal chromosome number 20 and a population doubling time of 10 to 14 hours was used.

This cell line is capable of developing resistance to 6-thioguanine (6TG) resulting from lack of hypoxanthine guanine phosphoribosyl transferase (hprt) enzyme activity as a result of mutation at the X chromosomes.

This cell line was screened for the absence of mycoplasma contamination and was certified free of mycoplasma contamination on 08 December 2015.

Cells were grown in tissue culture flasks at 37 ± 1 °C in a humidified carbon dioxide incubator (5 ± 0.2 % CO2 in air)

Evaluation criteria:

When all the validity criteria are fulfilled:

1. A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
• At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• The increase is concentration-dependent when evaluated with an appropriate trend test
• Any of the results are outside the distribution of the historical vehicle control data

When all of these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.



2. A test chemical is considered to be clearly negative if, in all experimental conditions examined:
• None of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
• There is no concentration-related increase when evaluated with an appropriate trend test
• All results are inside the distribution of the historical vehicle control data

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.


3. There is no requirement for verification of a clearly positive or negative response.


4. The results will be considered equivocal if they do not meet all the criteria specified for a positive or negative response respectively.

Statistics:

A power transformation procedure (Snee and Irr, 1981) was used with which, the observed mutant frequency was transformed using the formula:

Y = (X + A) B

where,
Y=transformed mutant frequency
X=observed mutant frequency
and A, B=constants.

Statistical analysis of the experimental data was carried out using validated copies of SYSTAT Statistical package version 12.0. In cases where analysis of variance was significant at p < 0.05, a Dunnett’s test was conducted, comparing each treatment group and the positive control to the vehicle control (p < 0.05).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

RESULTS

S9 Characterization

a. Sterility Check

The S9 homogenate was found to be sterile.

b. Metabolic Activation

The S9 homogenate was found to be active as evidenced by its ability to metabolize the promutagens, 2-Aminoanthacene and Benzo(a) pyrene to mutagens using S. typhimurium strain TA 100.

c. Protein Content

The protein content of the S9 homogenate was 29.5 mg/mL.

Preliminary Solubility Test

Hostaperm-Orange GR formed a free flowing suspension in DMSO at 50000 µg/mL. DMSO is one of the organic vehicles compatible with this test system. Hence DMSO was selected as the vehicle for the gene mutation assay.

Preliminary Cytotoxicity Test and Justification for the Selection of Target Top Concentration

At the end of 3-hour exposure to treatment, Hostaperm-Orange GR did not precipitate in the test medium at any of the tested concentrations.

At the end of 3-hour exposure to treatment, pH of the test medium in the presence of metabolic activation ranged from 6.98 to 7.10 with 7.02 in the DMSO control while in the absence of metabolic activation it was between 6.99 and 7.10 with 6.98 in the DMSO control.

At the end of 3-hour exposure to treatment, osmolality of the test medium at the highest test item treatment as well as highest soluble level (500 µg/mL) was 0.455 and 0.450 OSMOL/kg, in the presence and absence of metabolic activation, respectively, while the corresponding osmolality in the DMSO control was 0.455 and 0.446 OSMO L/kg in the presence and absence of metabolic activation, respectively.

Hostaperm-Orange GR did not show evidence of significant cell growth inhibition as Relative Survival (10 to 20% RS compared to vehicle control) up to 40 µg/mL in the presence as well as in the absence of metabolic activation.

In the presence of metabolic activation, at and above 80 µg/mL, there were insufficient cells and also the density of damaged cells was more, hence the cells were not processed for estimation of RS. In the absence of metabolic activation, at 80 and 160 µg/mL, there were insufficient cells and also the density of damaged cells was more, hence the cells were not processed for estimation of RS. At and above 320 µg/mL, there was no cell monolayer.

Based on these observations a maximum of 50 µg/mL was tested in the gene mutation assay.

Gene Mutation Assay

Parallel cytotoxicity test results are presented in Tables 4 and 5 for Experiments 1 and 2, respectively. Summary results of gene mutation assay are presented in Tables 6 and 7 for Experiments 1 and 2, respectively.

There was no evidence of excessive cytotoxicity (i.e.,<10 % RS) at any of the tested concentrations either in the presence or absence of metabolic activation. The RS values in the presence of metabolic activation, ranged from 88 to 35% while in the absence of metabolic activation, ranged from 80 to 27% compared to the vehicle control.

The frequency of mutants in the DMSO control was within the range of the in-house historical control data (Annexure 2).

The test item did not cause a significant increase in the frequencies of mutants compared to the vehicle control in the presence or absence of metabolic activation at any of the tested concentrations.

Under similar conditions the positive control 3-methylcholanthrene (3-MCA) induced statistically significant increases in the mutant frequency as compared with the vehicle control.

DISCUSSION

The assay conditions fulfilled all the specifications as per the OECD 476 guideline.

Since, the test item exhibited cytotoxicity, test concentrations up to a maximum of 50 µg/mL were exposed in the assay in the presence and absence of metabolic activation, which met the maximum limit of exposure in case the test item is cytotoxic.

Similarly, all the acceptance criteria for a valid test were met.

The Adjusted Cloning Efficiency of the vehicle controls was ≥ 61%. The mean mutant frequency of the vehicle controls in each experiment was between 12.95 and 8.41 mutants per 106 clonable cells.

In experiment in the presence of metabolic activation, the positive control chemical produced a statistically significant increase in the mutant frequencies, demonstrating both the activity of the metabolic activation system and the responsiveness of the test system.

No evidence for the induction of gene mutation was observed in any of the concentrations of the test item either in the presence or in the absence of metabolic activation.

Taken together, the results indicated that Hostaperm-Orange GR does not have the potential to induce gene mutation either in the presence or in the absence of metabolic activation.

Conclusions:

It is concluded that the test item does not have the potential to induce gene mutation in CHO-K1 cells at the tested concentrations and under the conditions of testing employed.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Executive summary:

The genotoxic potential of the test item to induce gene mutation in mammalian cells was evaluated according to OECD TG 476 using Chinese Hamster ovary (CHO) cells.

 

The study consisted of a preliminary cytotoxicity test and a definitive gene mutation test. The gene mutation test comprised of two independent experiments, one each in the presence and absence of metabolic activation system (S9 fraction prepared from Aroclor 1254 induced rat liver).

 

Hostaperm-Orange GR formed a free flowing suspension in Dimethyl sulphoxide (DMSO) at 50000 µg/mL.

 

In a preliminary cytotoxicity test for the selection of test concentrations for the gene mutation assay, the test item did not show evidence of significant cell growth inhibition as Relative Survival (RS) up to 40 µg/mL in the presence as well as in the absence of metabolic activation. In the presence of metabolic activation, at and above 80 µg/mL, there were insufficient cells and also the density of damaged cells was more, hence the cells were not processed for estimation of RS. In the absence of metabolic activation, at 80 and 160 µg/mL, there were insufficient cells and also the density of damaged cells was more, hence the cells were not processed for estimation of RS. At and above 320 µg/mL, there was no cell monolayer. The test item did not precipitate in the test medium and did not show any appreciable change in the pH and osmolality of test medium. Based on these observations a maximum of 50 µg/mL was tested in the gene mutation assay. 

 

In the gene mutation test, CHO-K1 cells were exposed to the test item in duplicate at concentrations of 2, 6, 17 and 50 µg/mL of the medium for 3 hours in the presence (Experiment 1) and absence (Experiment 2) of metabolic activation. In a similar way, a concurrent vehicle control (DMSO) and a positive control, 3-methylcholanthrene (Experiment 1) was also tested in duplicate.

 

There was no evidence of induction of gene mutations in any of the test item treated cultures either in the presence or absence of metabolic activation. In each of these experiments, the respective positive controls produced a statistically significant increase in the frequencies of mutants, under identical conditions.

The results of the forward gene mutation test at the hprt locus indicated that the test item was non-mutagenic under the conditions of this study.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

no data available

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Pigment Orange 43 did not reveal any mutagenic effect in the

• bacterial reverse mutation assay (AMES) in the presence or absence of metabolic activation,
• in the in vitro gene mutation study in mammalian cells (HPRT) in the presence or absence of metabolic activation, and
• in the mammalian cell chromosome aberration test in the presence or absence of metabolic activation.

Based on these findings the test item has not to be classified as germ cell mutagen according to Regulation (EC) No 1272/2008.