Calcium 4-[(5-chloro-4-methyl-2-sulphonatophenyl)azo]-3-hydroxy-2-naphthoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Red 48:2 is not mutagenic in bacteria as assessed both with a standard Ames test (Fouillet 1978) and the modified Prival assay (Sokolowski 2007). It is also not mutagenic in mammalian cells in vitro (Wollny 2007). It is not clastogenic in vitro (Höpker 2007) and it does not induce unscheduled DNA synthesis in human fibroblasts and primary hepatocytes from arochlor 1254-induced rats (Puri 1985).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

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 (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Identity: TKA10334
- Aggregate state at room temperature: solid
- Colour: red
- Stability in solvent: Not indicated by the sponsor
- Storage: room temperature
- Expiration Date: January 30, 2009
- Batch No.: non-surface treated PR48:2
- Content: 99.4 %

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I
Exposure 4h, preparation interval 18h, with and without S9-mix: 0.6, 1.3, 2.5, 5, 10 and 20 microgramm/plate

Experiment II
Exposure 18h, preparation interval 18h, without S9-mix: 2.8, 6.6, 11.3, 22.5, 45.0, 90, 180 and 360 microgramm/plate
Exposure 28h, preparation interval 28h, without S9-mix: 2.8, 6.6, 11.3, 22.5, 45.0, 90, 180 and 360 microgramm/plate
Exposure 4h, preparation interval 28h, with S9-mix: 0.7, 1.4, 2.8, 6.6, 11.3 and 22.5 microgramm/plate

Vehicle / solvent:

deionized water (suspension)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: ethylmethane sulfonate

Details on test system and experimental conditions:

On the day of the experiment (immediately before treatment), the test item was suspended in deionised water. The final concentration of deionised water in the culture medium was 10 % (v/v). The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

A pre-test on cell grovth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item (2). Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - ViO. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used: In a quantitative assessment, exponentially growing cell cultures (seeding about 40,000 cells/ slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. The cells were stained 24 hrs after start of treatment. Using a 400-fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group). The cell number of the treatment groups is given as % cells in relation to the control. The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 microL/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible. With respect to the ability to formulate a homogeneous suspension of TKA 10334, in the pretest, 720 microgramm/mL was applied as top concentration for treatment of the cultures. Test item concentrations between 5.6 and 720 microgramm/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item, after 4 hrs treatment, was observed at 5.6 microgramm/mL and above. Since no relevant toxicity was observed up to the highest applied and/or scorable concentration in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, for Experiment I, in the absence and the presence of S9 mix, 20 microgramm/mL was chosen as top treatment concentration. Dose selection of Experiment II, was also influenced by test item toxicity. In the range finding experiment, cleariy reduced cell numbers were observed, after 24 hrs exposure, with 180 microgramm/mL and above. Therefore, 360 pg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix, 22.5 microgramm/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.

Evaluation criteria:

The chromosome aberration test performed in our laboratory is considered acceptable if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory's historical control data range: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory's historical control data.

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all scored dose groups is in the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps); and/or
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps); and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed. Statistical significance was confirmed by means of the Fisher's exact test (9) (p < 0.05).

However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not cleariy met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed. Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid, a test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory's historical control data range (0.0 - 5.2 % polyploid cells).

Statistics:

not applicable

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In a range finding pre-test on toxicity, cell numbers were scored 24 hrs after start of treatment as an indicator for cytotoxicity. Concentrations between 5.6 and 720 microgramm/mL were applied. In the absence of S9 mix, no toxic effects were observed up to the highest applied concentration. In the presence of S9 mix, no toxic effects could be observed up to the highest scorable concentration. In contrast, 24 hrs continuous treatment with 180 microgramm/mL and above in the absence of S9 mix induced strong toxic effects.

In the pre-experiment, in the absence and presence of S9 mix, precipitation of the test item in culture medium was observed after treatment with 5.6 microgramm/mL and above. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 277 mOsm, pH 7.5 versus 274 mOsm and pH 7.5 at 720 microgramm/mL).

In Experiment I, in the absence and presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 10 microgramm/mL. In Experiment II, in the absence and presence of S9 mix, precipitation occurred after treatment 5.6 microgramm/mL and above.

In Experiment I, in the absence and presence of S9 mix, and in Experiment II, in the presence of S9 mix, no toxic effects indicated by reduced mitotic indices and/or reduced cell numbers of below 50 % of control were observed after treatment up to the highest applied test item concentration. In Experiment II, in the absence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, being far in excess of test item precipitation. In both experiments, in the absence and presence of S9 mix, no statistically significant or biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed.

The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, excluding gaps) and within the range of the laboratory's historical control data range: 0.0-4.0 % aberrant cells, excluding gaps. In Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, the number of aberrant cells, excluding gaps, was increased in a dose-related manner. Since all values were cleariy within the range of the laboratory's historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps), this observation has to be regarded as biologically irrelevant.

In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.4-3.1 %) as compared to the rates of the solvent controls (2.2 - 3.0 %).

In both experiments, either EMS (500 or 900 microgramm/mL) or CPA (1.4 or 2.0 microgramm/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

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: mammalian cell gene mutation assay

Specific details on test material used for the study:

- Identity: TKA10334
- Aggregate state at room temperature: solid
- Colour: red
- Stability in solvent: Not indicated by the sponsor
- Storage: room temperature
- Expiration Date: January 30, 2009
- Batch No.: non-surface treated PR48:2
- Content: 99.4 %

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-napthoflavone induced rat liver S9

Test concentrations with justification for top dose:

EXPERIMENT I
- 4h treatment, with and without S9-mix: 11.3, 22.5, 45, 90, 180 and 720 µg/plate (precipitation at 90, 180 and 720 µg/plate)

Experiment II
- 4h treatment, with S9-mix: 5.6 11.3, 22.5, 45, 90 and 720 µg/plate (precipitation at abd above 22.5 µg/plate)
- 24h treatment, without S9-mix: 5.6 11.3, 22.5, 45, 360 and 720 µg/plate (precipitation at and above 45 µg/plate)

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was suspended in deionized water. The final concentration of deion. water in the culture medium was 10 % v/v.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

other: ethylmethane sulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 4h in serum-free medium or 24h in growth medium
- Expression time (cells in growth medium): 7 days for cloning efficiency, 10 days for expression
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT: 6-Thioguanine

NUMBER OF REPLICATIONS: 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10® cells found in the negative and/or solvent controls fall within the laboratory historical control data range of 2001 - 2006.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the negative and/or solvent controls must exceed 50 %.

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system. A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 31.8 mutants per 10® cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of negative and solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The number of mutant colonies obtained in the groups treated with the test item was compared to the solvent control groups.
A trend is judged as significant whenever the p-value (probability value) is below 0.05.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006 - 2007

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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Aggregate State at Room Temperature: Solid
- Colour: Red
- Stability in Solvent: > 72 hours in DMSO at room temperature > 72 hours in Sesame Oil at room temperature
- Storage: Room temperature
- Expiration Date: April 04, 2010
- Identity: Graphtol-Rot P2B VP 2747
- Batch No.: Vers. EB 98-04 or KRON 393014
- C.I. Pigment Red 48:2 84.8 % (w/w)
- C.I. Pigment Red 48:3 4.1 % (w/w)

Target gene:

histidine operon

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-Naphthoflavone induced rat liver S9 and S9 liver microsomal fraction prepared from the liver of 7 - 8 weeks old male Syrian golden hamsters

Test concentrations with justification for top dose:

- Pre-Experiment/Experiment I : 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
- Experiment II: 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

DMSO
The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

congo red

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-aminoanthracene

Remarks:

strains TA 100 and TA 1535 without S9

Details on test system and experimental conditions:

In the pre-experiment the concentration range of the test item was 3 - 5000 microgramm/plate. It was the plate-incorporation experiment. The pre-experiment is reported as experiment I since the criteria mentioned above were met and 5000 microgramm/plate were chosen as maximal concentration. Due to minor toxic effects and the precipitation of the test item in the pre-experiment, seven concentrations were tested in experiment II. Experiment II was performed with hamster liver S9-mix in the pre-incubation set-up.

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:

No statistical evaluation of the data was required.

Species / strain:

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

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:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was observed from 333 microgramm/plate up to 5000 microgramm/plate in the test tubes. Precipitation was also observed on the agar plates at 1000 and 2500 microgramm/plate without S9 mix and from 1000 to 5000 microgramm/plate with S9 mix in experiment I, and from 1000 microgramm/plate up to 5000 microgramm/plate with and without S9 mix in experiment II. The undissolved particles had no influence on the data recording.
The plates incubated with the test item showed normal background growth up to 5000 microgramms per plate with and without metabolic activation in both experiments. Minor toxic effects, evident as a reduction in the number of revertants (below a factor of 0.5), were observed in some strains at concentrations of 2500 microgramms per plate or higher.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro mutagenicity in bacteria

The mutagenicity in vitro was assessed in two tests applying both the standard assay with rat liver homogenate and the modified assay for azo compounds (Prival-assay). Tested samples were commercial products containing varying amounts of additives. With the limitation in the number of tester strains, the non-key study is reliable and valid in regard to design, positive and negative control and concentrations. The key study was performed following the latest OECD testing guideline (OECD 471, adopted July 12, 1997) and the principles of GLP and included the Prival assay modification to take into account reductive metabolism of the azo bond. 

In vitro mutagenicity in mammalian cells

Mutagenicity in mammalian cells in vitro was investigated in an HPRT test following OECD testing guideline 476 (adopted July 21, 1997) and the principles of GLP. The test was performed with a sample synthesized without additives and a purity of 99.4%. No indication of a mutagenic effect was observed at concentrations up to 720mg/plate, at least the two highest concentrations being in the precipitating range.This is consistent with the absence of unscheduled DNA synthesis as tested with both with human fibroblasts (Puri 1985) and with primary hepatocytes prepared from arochlor1254-induced rats (Puri 1985). The latter studies were performed under GLP and in combination fulfil the requirements of OECD testing guideline 482 (adopted October 23, 1986). For the DNA-repair assays, concentrations were in similar range and also resulted in precipitation. Higher concentrations were too toxic for scoring. For these two studies, commercial samples with adequately high pigment content were tested.

In vitro clastogenicity in mammalian cells

Clastogenicity in mammalian cells was investigated in a guideline (OECD 473. adopted July 21, 1997) and GLP compliant study (Höpker 2007). The test was performed with a sample synthesized without additives and a purity of 99.4%. The highest evaluable concentrations were chosen based on visual observation of precipitates. No indication of clastogenicity or polyploidy were observed in either study.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity according to Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008.