Registration Dossier

Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out
Special Brilliant Blue FFR [CAS no. 6505-30-2]

- Name of the substance for which the testing proposal will be used [if different from tested substance]
Not applicable; test shall be conducted with Special Brilliant Blue FFR [CAS no. 6505-30-2]

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
According to REACH regulation Annex VIII and IX Column 2 chapter 8.4 appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
Annex VIII; Column 2 chapter 8.4: “appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII”.
Annex IX Column 2 chapter 8.4: “[i]f there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant”.

The most recent ECHA position document 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)…
(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)

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.”
“The in vivo alkaline single cell gel electrophoresis assay, also called alkaline Comet Assay is a method measuring DNA strand breaks in eukaryotic cells (OECD TG 489)”.
“The mammalian in vivo micronucleus test is used for the detection of damage induced by the test substance to the chromosomes or the mitotic apparatus of erythroblasts, by analysis of erythrocytes as sampled in bone marrow and/or peripheral blood cells of animals, usually rodents (mice or rats). The purpose of the micronucleus test is to identify substances (liquid or solid) that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage (OECD TG 474).

Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) a combined micronucleus assay and alkaline comet assay in the rat is proposed to evaluate potential mutagenic activity in vivo. There is no in vitro cytogenicity test or in vitro gene mutation test in mammalian cells available. Based on animal welfare reasons a single, combined test is proposed to investigate potential genotoxicity in vivo.
The tests shall be conducted according to OECD TG 489 (in vivo mammalian alkaline comet assay) combined with OECD TG 474 (Mammalian erythrocyte micronucleus test) on rats by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/ jejunum and bone marrow. For the micronucleus test the bone marrow shall be analysed.

Special Brilliant Blue FFR is a crystalline, shiny metallic brown solid with a molecular weight of ca. 790 g/mol. The water solubility of Special Brilliant Blue FFR at 20 °C is determined to be at least 292 g/L; and the octanol-water coefficient is log Pow = -2.49 at 20°C. In the acute oral toxicity study the discriminating dose was 2500 mg/kg bw; no signs of intoxication were reported. Overall, in vivo some absorption after oral intake is assumed.
In the Ames test Special Brilliant Blue FFR [CAS no. 6505-30-2] was only positive in one bacterial strain, TA 98, and only in the presence of metabolic activation (S9 mix). Therefore some metabolism of Special Brilliant Blue FFR [CAS no. 6505-30-2] might be assumed in vivo and further in vitro testes are not proposed.


- Available GLP studies
There is no in vivo genotoxicity study conducted according to GLP available.

- Available non-GLP studies
There is no in vivo genotoxicity study available.

- Historical human data
No data available.

- (Q)SAR
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 VIII Column 2 chapter 8.4 appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VII.
Due to the positive results of the Ames test according to OECD Guideline 471 (Bacterial Reverse Mutation Assay Test) in Salmonella typhimurium strain TA 98 (with metabolic activation) a combined micronucleus assay (OECD TG 474) and alkaline comet assay (OECD TG 489) in the rat is proposed to evaluate potential mutagenic activity in vivo. There is no in vitro cytogenicity test or in vitro gene mutation test in mammalian cells available. Based on animal welfare reasons a single, combined test is proposed to investigate potential genotoxicity in vivo.

- Weight of evidence
According to REACH regulation Annex VIII Column 2 chapter 8.4 appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
The data from the available in vitro genotoxicity assays is not sufficient for a weight of evidence consideration according to the ECHA’s REACH guidance documents.

- Grouping and read-across
Based on the chemical structure and the physicochemical characteristics of Special Brilliant Blue FFR [CAS no. 6505-30-2] no grouping or read-across approach was identified to fill the information for an in vivo mutagenicity study according to Annex VIII column 2 chapter 8.4.

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

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

- Other reasons [if applicable]
According to REACH regulation Annex VIII Column 2 chapter 8.4 appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.

The most recent ECHA position document 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)…
(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)

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.”

In the Ames test Special Brilliant Blue FFR [CAS no. 6505-30-2] was only positive in one bacterial strain, TA 98, and only in the presence of metabolic activation ( S9 mix). There is no in vitro cytogenicity test or in vitro gene mutation test in mammalian cells available.
No separate micronucleus assay (OECD TG 474) and alkaline comet assay (OECD TG 489) are proposed. For animals welfare reasons the combination of the micronucleus assay (OECD TG 474) and alkaline comet assay (OECD TG 489) in the rat is proposed. Based on the combination of the two studies, a significant reduction in the number of animals used for the combined assay is obtained.


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:
According to REACH regulation Annex VIII and IX Column 2 chapter 8.4 appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
Annex VIII; Column 2 chapter 8.4: “appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII”.
Annex IX Column 2 chapter 8.4: “[i]f there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant”.


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) a combined micronucleus assay and alkaline comet assay in the rat is proposed to evaluate potential mutagenic activity in vivo.

Special Brilliant Blue FFR is a crystalline, shiny metallic brown solid with a molecular weight of ca. 790 g/mol. The water solubility of Special Brilliant Blue FFR at 20 °C is determined to be at least 292 g/L; and the octanol-water coefficient is log Pow = -2.49 at 20°C. In the acute oral toxicity study the discriminating dose was 2500 mg/kg bw; no signs of intoxication were reported. Overall, in vivo some absorption after oral intake is assumed.
In the Ames test Special Brilliant Blue FFR [CAS no. 6505-30-2] was only positive in one bacterial strain, TA 98, and only in the presence of metabolic activation (S9 mix). Therefore some metabolism of Special Brilliant Blue FFR [CAS no. 6505-30-2] might be assumed in vivo and further in vitro testes are not proposed.

The tests shall be conducted according to OECD TG 489 (in vivo mammalian alkaline comet assay) combined with OECD TG 474 (Mammalian erythrocyte micronucleus test) on rats by the oral route (gavage). The following systemic and local organs shall be analysed: liver, glandular stomach, duodenum/ jejunum and bone marrow. For the micronucleus test the bone marrow shall be analysed.

Data source

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Results and discussion

Applicant's summary and conclusion