Strontium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Pigment Red 57(Sr) is not genotoxic as assessed both with standard Ames tests and the modified Prival assay (OECD 471, GLP, BASF 1992 and DIC 2005) and with the chromosome aberration study in vitro (OECD 473, GLP, DIC 2005). Furthermore, the analogue Ca-salt Pigment Red 57:1(Ca) is not mutagenic in mammalian cells in vitro (OECD 476, GLP, Wollny 2008) and does not induce unscheduled DNA synthesis in human fibroblasts and primary hepatocytes from arochlor 1253-induced rats (OECD 482, GLP, Hertner 1987 and Meyer 1987).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

Please see the category read-across justification in the category object.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Highest tested concentration caused precipitation but was less than 5 mg/plate. Both experiments performed with pre-incuation method.

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:

- Name of test material (as cited in study report): Strontium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo}-2-naphthoate
- Substance type: red pigment
- Physical state: solid


Degree of solubility
Water: <50 mg/mL (measured at Hita Laboratory)
DMSO: <50 mg/mL (measured at Hita Laboratory)

- Other: Supplier DAINIPPON INK AND CHEMICALS, INCORPORATED

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 and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Range-finding test with all strains: 4.88, 19.5, 78.8, 313, 1250 and 5000 μg/plate
- Main test 1 and 2: 7.88, 19.5, 39.1, 78.1, 156 and 313 μg/plate

Vehicle / solvent:

- Vehicle: DMSO; the test substance was insoluble in distilled water at 50 mg/mL and it was a good suspension in DMSO at 50 mg/mL. The test substance suspension of 50 mg/mL prepared with DMSO was considered to be stable there from the facts being neither any change in color nor heating at room temperature within 2 hours after preparation. Therefore, DMSO was preferably selected as a solvent.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide & 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine dihydrochlorid

Details on test system and experimental conditions:

Amino acid requirement, sensitivity to ultraviolet-rays, rfa membrane mutation, presence or absence of plasmid pKM101 and negative and positive control values of test strains were examined. Spectrophotometric grade of dimethyl sulfoxide was added to fresh overnight cultures of the test strains which had been confirmed to have these properties at a ratio of 0.9:10. The mixture was frozen as a stock culture in an ultradeep freezer below -80°C.

Evaluation criteria:

The test substance was judged positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner and a reproducibility of the test results was also assured. In all other cases, it was judged negative.

Statistics:

Any statistical methods were not used.

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

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:

No genotoxicity was observed in the range-finder test that was performed in all strains with concentrations up to 5 mg/plate. Precipitation was observed at concentrations of 313 μg/plate and above.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Osmolarity not measured.

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:

- Name of test material (as cited in study report): Strontium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo}-2-naphthoate
- Substance type: red pigment
- Physical state: solid


Degree of solubility
Water: <50 mg/mL (measured at Hita Laboratory)
DMSO: <50 mg/mL (measured at Hita Laboratory)
Acetone < 472 mg/mL (measured at Hita Laboratory)

- Other: Supplier DAINIPPON INK AND CHEMICALS, INCORPORATED

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster lung fibroblast CHL/IU

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and 5,6-benzoflavone induced rat liver S9

Test concentrations with justification for top dose:

- Short-term treatment without S9 mix: 75, 150, 300, 600, 1200 and 2400 μg/ml
- Short-term treatment with S9 mix: 37.5, 75, 150, 300, 600, 1200 and 2400 μg/ml
- Continuous treatment, no S9 mix: 26.3, 39.5, 59.3, 88.9, 133 and 200 μg/ml

In the range-finder studies, precipitation of the pigment and red discoloration of the medium was observed even at the lowest dose of 18.4 μg/ml both in the absence and presence of S9 mix, both at the 6h and 24h treatments.

Vehicle / solvent:

0.5% carboxymethylcellulose in water; this formed a more homogenous suspenstion than DMSO and acetone.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6h (short) or 24h (continous)
- Fixation time (start of exposure up to fixation or harvest of cells): 24h


SPINDLE INHIBITOR (cytogenetic assays): Demecolcin (2h prior to fixation)
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: Duplicates


NUMBER OF CELLS EVALUATED: 200 metaphases per dose


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (cell counting after trypsination using a Microcell counter)


OTHER EXAMINATIONS:
- Determination of polyploidy: yes (Cells with more than 38 chromosomes)


OTHER: Cells were free of mycoplasma, had 25 chromosomes per cell and a population doubling time of about 15h. The incidence of spontaneous chromosomes was less than 5%.

Evaluation criteria:

- Cells with more than 38 chromosomes were judged as having a numerical aberration.
- A rate of more than 10% of cells with numerical aberrations was counted as positive.
- A rate of more than 5% structural aberrations was counted as positive.
- Effects had to show a dose-response relationship.

Statistics:

Any statistical methods were not used.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

other: clastogenicity negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

other: polyploidy positive at precipitating and cytotoxic concentrations.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

not applicable

Additional information on results:

IC50 (24h) = 75 μg/mL
IC50 (6h, without S9 mix) = 240 μg/mL
IC50 (6h, with S9 mix) = 480 μg/mL

Precipitation of the test item was observed at all concentrations at the start and at the beginning of the treatment incubation and also at the end of the culture. In the range-finder studies, precipitation of the pigment and red discoloration of the medium was observed even at the lowest dose of 18.4 μg/ml both in the absence and presence of S9 mix, both at the 6h and 24h treatments.
Measurement of pH confirmed that medium discoloration was no effect of a change in pH.

- Numerical abberrations occured in the absence of S9 mix after 6h treatment: incidence of 16.5, 18.5 and 34% at 75, 150 and 300 μg/mL, respectively.

- Numerical abberrations occured in the presence of S9 mix after 6h treatment: incidence of 5.5, 11 and 14% at 150, 300 and 600 μg/mL, respectively.

- Numerical abberrations occured after 24h treatment: incidence of 17, 21 and 26 % at 39.5, 59.3 and 88.9 μg/mL, respectively.

The numerical aberrations are clearly above the incidence of the historical control data given in the attachment of the study report. No increase in numerical aberrations were noted in the range-finder studies at 18.4 μg/ml both in the absence and presence of S9 mix

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). The studies were performed following the latest OECD testing guideline (OECD 471) and the principles of GLP. The standard test was performed with doses of 7.88, 19.5, 39.1, 78.1, 156 and 313 μg/plate using the pre-incubation method and DMSO as vehicle, and included all five tester strains (DIC 2005a). The assay with the Prival modification was performed with Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and doses of 0, 20, 100, 500, 2500 and 5000 μg/plate in DMSO were applied (BASF AG 1992).

 

In vitro mutagenicity in mammalian cells

Mutagenicity in mammalian cells in vitro was investigated with the calcium analogue Pigment Red 57:1(Ca) in an hprt test following OECD testing guideline 476 (adopted July 21, 1997) and the principles of GLP. No indication of a mutagenic effect was observed at concentrations up to 1000 µg/mL, the two highest concentrations being in the precipitating range. This is consistent with the absence of unscheduled DNA synthesis of the calcium analogue as tested with both with human fibroblasts (Meyer 1987) and with primary hepatocytes prepared from Arochlor1254-induced rats (Hertner 1987). The latter studies were performed under GLP and in combination fulfil the requirements of OECD testing guideline 482 (adopted October 23, 19869. For the DNA-repair assays, concentrations were in similar range and also resulted in precipitation. Higher concentrations were too toxic for scoring. For all three studies, commercial samples of known composition were tested.

 

In vitro clastogenicity in mammalian cells

For clastogenicity in vitro, experimental data from studies with samples of Pigment Red 57(Sr) and the analogues Pigment Red 57:1(Ca) and 63:3(Sr) were taken into account.

 

For Pigment Red 57(Sr), clastogenicity in mammalian cells was investigated in a guideline (OECD 473. adopted July 21, 1997) and GLP compliant study (DIC 2005). Purity of the test sample is adequate. Pigment Red 57-Sr was applied as a suspension in 0.5% carboxymethylcellulose in water. Precipitation of the test item was observed at all concentrations at the start and at the beginning of the incubation and the test item coloured the culture medium red. In the range-finding test concentrations as low as 18.5 μg/mL caused precipitation as well as culture medium colouration. For the main study, the highest dose was chosen based on a reduction in growth rate by less than 50%, and so all doses were in the precipitating range. A dose-dependent increase in numerical aberrations occurred in the absence and presence of S9 mix after 6h and 24h treatment in parallel to cytotoxicity. This increase was above the historical control range and could not be attributed to a change in pH. Changes in osmolality also cause chromosome breaks in cell culture studies, but osmolality was not measured in this study. After 24h treatment, the incidence of numerical aberrations was 17, 21 and 26 % at 39.5, 59.3 and 88.9 μg/mL, respectively. Higher concentrations could not be scored because the precipitates were too numerous. In the presence of S9 mix after 6h treatment, the incidence was 5.5, 11 and 14% at 150, 300 and 600 μg/mL, respectively. In the absence of S9 mix after 6h treatment, the incidence was 16.5, 18.5 and 34% at 75, 150 and 300 μg/mL, respectively. As cultured cells are sensitive to mechanical effects of precipitates and no doses in the non-precipitating range were tested, the relevance of the increase in polyploidy index is questionable. A concomitant increase in cytotoxicity and polyploidy index indicates that the actual pigment component is very unlikely to be the cause.

 

Experimental data on chromosome aberration in vitro was available for a related strontium BONA metal laked pigment Pigment Red 63:3(Sr) and the calcium analogue Pigment Red 57:1(Ca). The strontium analogue Pigment Red 63:3(Sr) (3-hydroxy-4-[(1-sulfonato-2-naphthyl)azo]-2-naphthoate) contains a sulfonated naphthyl ring instead of a sulfonated phenyl ring. A GLP and OECD guideline 473 compliant study showed absence of structural aberrations for both Pigment Red 57:1(Ca) (Hoffmann 2008 and MHLW 1993) and 63:3(Sr) (DIC 2008). As determined in the course of the genotoxicity tests, Pigment Red 63:3(Sr) is of similar low solubility in water and organic solvents as Pigment Red 57(Sr), and precipitation in the cell culture medium also occurred at concentrations as low as 19.9 μg/mL. In contrast to the test with Pigment Red 57(Sr), no significant reduction in growth rate was observed despite the precipitation and so the highest applied concentrations was 5000 μg/mL. The tested sample had an adequately high purity. No increase in polyploidy index was observed for the more soluble calcium salt analogue Pigment Red 57:1(Ca). The highest evaluable concentrations were chosen based on visual observation of precipitates which occurred at 250 μg/ml and above. No indication of clastogenicity or polyploidy were observed in either study. For both studies, commercial samples of known composition were tested. In one study, 0.5% carboxymethylcellulose in water was used as vehicle, whereas the other used DMSO. Whereas precipitation occurred at some point for all three pigment samples, concomitant cytotoxicity as indicated by inhibition of growth rate was only observed with the sample of Pigment Red 57(Sr). It is expected that any potential effect of strontium would be similar for Pigment Red 57(Sr) and 63:3(Sr). Therefore, the polyploidy increase is not considered to be related to Sr. As the organic part of Pigment Red 57:1(Ca) and 57(Sr) is identical, and Pigment Red 57:1(Ca) showed less precipitation, also no hazardous properties are attributed to the organic part. It is speculated that cytotoxicity and polyploidy are either related to differences in the crystal form, osmolality or to an unidentified impurity in the test sample. As the increase in polyploidy occurred irrespective of metabolic activation, it is more likely to be an artifact.

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.