pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study planned

Remarks:

In Vivo Mammalian Alkaline Comet Assay according to OECD TG 489

Study period:

An In Vivo Mammalian Alkaline Comet Assay is proposed. The test period will be established once the ECHA approves this Testing Proposal.
The test shall be conducted according to OECD TG 489 on rat and by the oral route (gavage). The following systemic and

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS
In Vivo Mammalian Alkaline Comet Assay (OECD 489) with the registered substance.

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Bayscript Black TP LXS 51134 (pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate; CAS No. 2259360-19-3)
- Name of the substance for which the testing proposal will be used [if different from tested substance:
not applicable


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
According to REACH regulation Annex VII Column 2 chapter 8.4. further mutagenicity studies shall be considered in case of a positive result.
The ECHA position document ‘Three recently approved in vivo genotoxicity test guidelines’ (Revised in February 2018) on in vivo genotoxicity testing indicates:
“When there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD TG 471) or from an in vitro gene mutation study in mammalian cells (OECD TG 476 for tests using the Hprt and xprt genes; OECD TG 490 for tests using the thymidine kinase gene), adequate somatic cell in vivo tests to investigate gene mutations are TGR (OECD TG 488), comet assay (OECD TG 489) or, if justified, Unscheduled DNA Synthesis (UDS) test with mammalian liver cells in vivo (OECD TG 486)… Consequently, a testing proposal must be submitted for in vivo tests intended to meet the information requirements of the REACH Regulation. Following examination of such a testing proposal, ECHA has to approve the test in its evaluation decision before it can be undertaken.” (Cited from ECHA position document “Three recently approved in vivo genotoxicity test guidelines (Revised in February 2018). https://echa.europa.eu/documents/10162/21650280/oecd_test_guidelines_genotoxicity_en.pdf)

An Ames test was performed to investigate the potential of Bayscript Black TP LXS 51134 (pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate; CAS No. 2259360-19-3) to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471.
Because the test item is an azo-dye, a special test design according to Prival and Mitchell (1982) was applied as proposed in OECD TG 471. The assay was performed in three independent experiments. Experiment I was performed with induced rat liver S9 mix as an exogenous metabolic activation system and experiment II and IIa were performed with non-induced hamster liver S9 mix.
During the mutagenicity test and under the experimental conditions reported (OECD TG 471, modified version for azo-dyes), the test item did induce gene mutations by frameshifts in the genome of the strain TA 98 in the presence of non-induced hamster liver S9 mix.
Therefore, Bayscript Black TP LXS 51134 is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.
Further in-vitro or in-vivo genetic toxicity tests are not available.
Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) the conduction of an in-vivo Comet assay is proposed to evaluate potential mutagenic activity in vivo.
Acute oral toxicity: The registered substance was tested at a starting dose of 300 mg/kg bw and at 2000 mg/kg bw in the main study of an acute oral toxicity study in female Wistar rats following OECD TG 420. One animal of the main test treated with 2000 mg/kg body weight was euthanized on day 1 after application due to deterioration of clinical signs that were, if compared to the symptoms observed in the four other animals dosed with 2000 mg/kg b.w., unexpectedly severe. Therefore, it is possible that these individual symptoms were incidental. Clinical signs in the other 4 animals included secretion of Harderian glands, and staining by the couloured test item of fur, mouth, skin, urine and faeces. The acute median lethal oral dose (LD50) was demonstrated to be > 2000 mg/kg bw.
The results of this study indicate some systemic bioavailability of the test substance at very high doses (acute oral toxicity > 2000 mg/kg bw).

- Available GLP studies
There are no GLP-compliant in vivo genotoxicity studies available.

- Available non-GLP studies
There are no non-GLP-compliant in vivo genotoxicity studies available.

- Historical human data
No data available

- (Q)SAR
The QSAR Toolbox 4.4 identified no DNA alerts for Bayscript Black TP LXS 51134 regarding the mutagenicity tests AMES, CA (chromosomal aberration) and MNT (micronucleus test) by OASIS (endpoint specific profilers). However, in vitro (Ames test) and in vivo (Micronucleus) alerts were identified by ISS due to the aromatic diazo structure of Bayscript Black TP LXS 51134.
There is no QSAR model available which is accepted by ECHA for the endpoint in vivo genotoxicity in case of a positive in vitro genotoxicity study.

- In vitro methods
According to REACH regulation Annex VII Column 2 chapter 8.4. further mutagenicity studies shall be considered in case of a positive result.

The ECHA position document ‘Three recently approved in vivo genotoxicity test guidelines’ (Revised in February 2018) on in vivo genotoxicity testing indicates: “When there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD TG 471) or from an in vitro gene mutation study in mammalian cells (OECD TG 476 for tests using the Hprt and xprt genes; OECD TG 490 for tests using the thymidine kinase gene), adequate somatic cell in vivo tests to investigate gene mutations are TGR (OECD TG 488), comet assay (OECD TG 489) or, if justified, Unscheduled DNA Synthesis (UDS) test with mammalian liver cells in vivo (OECD TG 486)… Consequently, a testing proposal must be submitted for in vivo tests intended to meet the information requirements of the REACH Regulation. Following examination of such a testing proposal, ECHA has to approve the test in its evaluation decision before it can be undertaken.” (Cited from ECHA position document “Three recently approved in vivo genotoxicity test guidelines (Revised in February 2018).

Based on this ECHA document the conduction of an In Vivo Mammalian Alkaline Comet Assay (OECD 489) with Bayscript Black TP LXS 51134 (pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate; CAS No. 2259360-19-3) is proposed to evaluate potential mutagenic activity in vivo.

- Weight of evidence
According to REACH regulation Annex VII Column 2 chapter 8.4. further mutagenicity studies shall be considered in case of a positive result.
The data from the available in vitro genotoxicity assay (Ames test) is not sufficient for a weight of evidence consideration according to the ECHA’s REACH guidance documents. Further in-vitro or in-vivo genetic toxicity tests are not available.

- Grouping and read-across
Based on the chemical structure no grouping or read-across approach was identified to fill the default information for an in vivo mutagenicity study.

- Substance-tailored exposure driven testing [if applicable]
Not applicable

- Approaches in addition to above [if applicable]
Not applicable

- Other reasons:
The most recent ECHA position document ‘Three recently approved in vivo genotoxicity test guidelines’ (Revised in February 2018) on in vivo genotoxicity testing indicates: “When there is a positive result from an in vitro gene mutation study in bacteria (Ames test, OECD TG 471) or from an in vitro gene mutation study in mammalian cells (OECD TG 476 for tests using the Hprt and xprt genes; OECD TG 490 for tests using the thymidine kinase gene), adequate somatic cell in vivo tests to investigate gene mutations are TGR (OECD TG 488), comet assay (OECD TG 489) or, if justified, Unscheduled DNA Synthesis (UDS) test with mammalian liver cells in vivo (OECD TG 486)… Consequently, a testing proposal must be submitted for in vivo tests intended to meet the information requirements of the REACH Regulation. Following examination of such a testing proposal, ECHA has to approve the test in its evaluation decision before it can be undertaken.” (Cited from ECHA position document “Three recently approved in vivo genotoxicity test guidelines (Revised in February 2018).
https://echa.europa.eu/documents/10162/21650280/oecd_test_guidelines_genotoxicity_en.pdf)
Therefore the conduction of an In Vivo Mammalian Alkaline Comet Assay (OECD 489) with of Bayscript Black TP LXS 51134 (pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate; CAS No. 2259360-19-3) is proposed to evaluate potential mutagenic activity in vivo.
CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
There is no in-vivo study available to investigate gene mutations and the adaptation options as defined in Annexes VI to X (and column 2 thereof) are not applicable for this substance and this endpoint.
Therefore, as none of the general and specific adaptation rules in column 2 provide possibilities for omitting the testing and testing is technically feasible, there is no other option to formally fulfil the requirements as to propose an in vivo genetic toxicity assay. The conduction of an In Vivo Mammalian Alkaline Comet Assay (OECD 489) with Bayscript Black TP LXS 51134 (pentasodium 4-amino-3-[{4-[(2,4-disulfonatophenyl)diazenyl]-3-methylphenyl}diazenyl]-5-hydroxy-6-[(4-nitro-2-sulfonatophenyl)diazenyl]naphthalene-1,7-disulfonate; CAS No. 2259360-19-3) is proposed to fulfil the requirements of the ECHA position document ‘Three recently approved in vivo genotoxicity test guidelines’.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) the conduction of an in-vivo Comet assay According to OECD TG 489 is proposed to evaluate potential mutagenic activity in vivo.
The test shall be conducted according to OECD TG 489 on rats and by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/jejunum and bone marrow.

Materials and methods

Test guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Cas Number:

2259360-19-3

Molecular formula:

C29H17N8Na5O18S5

IUPAC Name:

pentasodium 4-amino-3-(2-{4-[2-(2,4-disulfonatophenyl)diazen-1-yl]-3-methylphenyl}diazen-1-yl)-5-hydroxy-6-[2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Administration / exposure

Route of administration:

oral: gavage