Calcium 4,5-dichloro-2-[[4,5-dihydro-3-methyl-5-oxo-1-(3-sulphonatophenyl)-1H-pyrazol-4-yl]azo]benzenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacteria

Read-across: OECD 471; GLP; S. typhimurium TA 1535, 1537, 98, and 100 and E. coli WP2 uvrA; with and without S9; 4 µg/plate - 5500 µg/plate; not mutagenic (2002)

Cytogenicity in mammalian cells

Read-across: OECD 473; GLP; Chinese hamster lung fibroblasts (V79); with and without S9; 25 – 500 µg/ml; clastogenic

Gene mutation in mammalian cells

Read-across: OECD 476; GLP; Chinese hamster ovary (CHO); with and without S9; 2.5 – 5200 µg/ml; not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read-across justification

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read-across justification

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Read-across: OECD 474; GLP; male mouse (NMRI); oral (gavage); 500, 1000, and 2000 mg/kg in a volume of 20 ml/kg; two administrations (24-h in-between); not clastogenic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See read-across justification

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Read across justification

There are only limited, non-reliable data available on genotoxicity of the test item. Therefore, it is acceptable to derive the information on mutagenicity and clastogenicity from experimental data of structural analogues (di-sodium and di-ammoinium salt) since all are salts with similar structures. In addition, the higher solubility and the lower molecular weight of the analogue substances give a safety margin

(a detailed read across justification is given in CSR, Annex I).

Performance and observations

There are four reliable, GLP-conform studies available to assess the potential of analogue substances to induce genotoxicity in vitro and in vivo.

The first study (BASF AG 2002b) was performed to investigate the potential of the test item (analogue calcium salt) to induce gene mutations according to the plate incorporation test using the S. typhimurium strains TA1535, TA 1537, TA 98, and TA 100, TA 1538 and the E. coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without S9 mix from aroclor induced rat liver and non-induced Syrian hamster liver. The test item, dissolved in DMSO, was tested at concentrations of 22 - 5500 µg/plate (standard test) and 4 - 2500 µg/plate (prival modification test). No substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation. Precipitation occured from 500 µg/plate onward.

In a second study (BASF AG 2002c), the test item (analogue calcium salt), dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells in two independent experiments. The experiments were carried out at 0 - 500 µg/plate, 4h exposure and with or without metabolic activation. The test article caused a significant, biological relevant increase in structural chromosomal aberrations in the presence of S-9 mix. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells and demonstrated the sensitivity of the test compound. Thus, the test substance is considered to be clastogenic under the in vitro conditions of this assay using V79 cells.

In addition to the chromosome aberration assay in vitro, an in vivo assay was conducted to evaluate the ability of the test article (analogue calcium/sodium salt 1:1) to induce micronuclei in bone marrow polychromatic erythrocytes in mice (Ciba 1996). Based on the results of the dose selection study, the maximum tolerated dose was estimated as >5000 mg/kg bw. In the micronucleus assay, the test article was suspended in 1.0% methyl cellulose (MC) and dosed by oral gavage at 1250, 2500, and 5000 mg/kg bw. Ten animals (five males and five females) were randomly assigned to each dose/harvest time group. Vehicle and positive control groups, euthanized approximately 24 hours after dosing, were included in the assay. The animals dosed with the test article were euthanized approximately 24, 48 and 72 hours after dosing for extraction of the bone marrow. The test material did not induce a significant increase in micronuclei in bone marrow polychromatic erythrocytes.

An Ames test was conducted to examine the mutagenic potential of the test substance in vitro (BASF AG 1982b). Salmonella strains TA 1535, 1537, 98 and 110 were used to determine the mutagenic potential at concetrations of 0 - 5000 microgramm/plate with and without meatbolic activation. The test material was a 10 : 1 mixture of the calcium and a corresponding sodium salt, dissolved in DMSO. An increase of revertant colonies after incubation in presence or absence of the substance was not observed. Although, the test item is an azo dye, a prival modification of the assay was not conducted. Thus, the results from this assay are not assignable.

At last, the substance (analogue diammonium salt) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (BASF 2011). Three experiments were carried out independently of each other at dose levels of 2.5 - 5200 ug/ml in absence and presence of a metabolic activation syten for 4h and 24h, respectively. Thereafter, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. Precipitation occurred from the lowest applied concentration onward. In all experimental parts in the absence of S9 mix cytotoxicity was observed at strongly precipitating concentrations only. The test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system. In the 1st Experiment after 4 hours exposure in the absence of S9 mix an increase in the number of mutant colonies was observed in a single culture which was not corroborated in the culture treated in parallel or in the repeat experiment (2nd Experiment).

Discussion

The test item as well as analogue substances (di-sodium and di-ammoinium salt) were tested for genotoxicity in vitro. The di-sodium salt was negative in a modified Ames test and positive for chromosomal aberrations in V79 cells. However, a micronulcei test in mice yielded a negative result. Structural chromosome aberrations in V79 cells may be explained by precipitation of the test item from 125 microgramm onward leading to cytotoxicity induced DNA-damages. Moreover, a description on analytical purity and impurities was not given. Thus, it is possible that the positive result might origine from impurities rather than from the test susbtance itself. The HPRT assay on a diammonium salt yielded a negative result for mutagenicity in mammalian cells. Therefore, the test substance is considered to be non-genotoxic.

Short description of key information:
There are only limited, non-reliable data available on genotoxicity of the test item. Thus, information from valid in vitro and in vivo studies of structural analogues was derived. In an Ames test conducted according to OECD 471 no genotoxicity was seen. In an in vitro chromosome aberration assay conducted according to OECD guideline 473 an increase in structural chromosome aberrations was seen, probably due to strong precipitation or impurities. An in vivo Micronucleus test according to OECD guideline 475 as well as an HPRT test on a strucutural analogue yielded negative results. Based on results of these studies the test substance was considered to be not genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

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 genotoxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Commission ERgulation (EU) 2018/1480 of Oct 4, 2018.