Registration Dossier

Diss Factsheets

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).
Justification for type of information:
Testing Proposal Comet assay, OECD 489 (6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione, CAS 21295-57-8), Macrolex Fluoreszenzrot 4B

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 local organs shall be analysed: liver, glandular stomach, duodenum/jejunum and bone marrow.

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).]
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:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8)
- Name of the substance for which the testing proposal will be used [if different from tested substance:
not applicable; test shall be conducted with 6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij] isoquinoline-2,7-dione (CAS 21295-57-8)



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 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)

An Ames test was performed to investigate the potential of 6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8) (Macrolex Fluoreszenzrot 4B) to induce gene mutations according to the plate incorporation test (experiment I and Ia) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA, following OECD TG 471. Under the experimental conditions of the test, Macrolex Fluoreszenzrot 4B did induce gene mutations by frameshifts in the genome of strain TA 1537 in the presence of S9 mix. Therefore, Macrolex Fluoreszenzrot 4B 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.
As described in IUCLID chapter 4 (physical and chemical properties), the water solubility of 6-(cyclo-hexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8) is < 4.3 µg/L, the log Pow is 4.56 and the particle size is 5.79 µm.
The registered substance was tested in a single dose of 2000 mg/kg bw in an acute oral toxicity study in female Wistar rats following OECD TG 420. There were no deaths during the study. As clinical signs hunched posture, stained faeces (dark red), decreased activity, fur stained by test item, piloerection, partially closed eyes, abdominal tension, secretion of Haderian glands, and nervousness were noted in the first two days after dosing. The animal showed expected gains in body weight over the observation period. Macroscopic examination at study termination on Day 14 revealed red to pink fur, congestion of the genitalia, enlarged heart, slightly blue coloured pharynx, slightly blue coloured front teeth of the lower jaw. The acute median lethal oral dose (LD50) to rats of Macrolex Fluoreszenzrot 4B was demonstrated in this study to be greater than 2000 mg/kg body weight.
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 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 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 of 6-(cyclo-hexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8) 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
The QSAR Toolbox 4.4 identified 2 analogous substances (3H-Naphtho[1,2,3-de]quinoline-2,7-dione, 3-cyclohexyl-6-(cyclohexylamino) (CAS 53949-78-3) and 3H-Naphtho[1,2,3-de]quinoline-2,7-dione, 6-(cyclohexylamino)-3-methyl- (CAS 71902-18-6. No relevant toxicological studies inclusive studies for genetic toxicity are available for the 2 analogous substances.
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 6-(cyclo-hexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8) 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 and in vivo genetic toxicity assay. The conduction of an In Vivo Mammalian Alkaline Comet Assay (OECD 489) with 6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione (CAS 21295-57-8) 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.

Data source

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Test material

Constituent 1
Chemical structure
Reference substance name:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
EC Number:
244-320-0
EC Name:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
Cas Number:
21295-57-8
Molecular formula:
C23H22N2O2
IUPAC Name:
6-(cyclohexylamino)-3-methyl-3H-dibenz[f,ij]isoquinoline-2,7-dione
Test material form:
solid: particulate/powder

Test animals

Species:
rat

Administration / exposure

Route of administration:
oral: gavage

Results and discussion

Applicant's summary and conclusion

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