3,4,5,6-tetrachloro-N-[2-(4,5,6,7-tetrachloro-2,3-dihydro-1,3-dioxo-1H-inden-2-yl)-8-quinolyl]phthalimide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No suitable data on genetic toxicity in vitro were available for the test substance. Therefore, read across on reliable studies from two analogue substances was performed.

 

- Ames test: read across to CAS 386254-45-1, according to OECD TG 471, GLP, negative

- Mouse lymphoma assay (MLA): read across to CAS 106276-80-6, according to OECD TG 476, GLP, negative

- Micronucleus test (MNT): read across to CAS 386254-45-1, according to OECD TG 487, GLP, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

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

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Reference 2

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

Justification for type of information:

Please see the attached justification.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Reference 3

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Preincubation test > 500 µg/plate; SPT at about 5500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No adequate data on genetic toxicity in vivo were available for the test substance. Therefore, read across on reliable studies from an analogue substance was performed.

 

- MNT: read across to CAS 106276-80-6, according to OECD TG 474, non-GLP, negative

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

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro:

Mutagenicity in bacteria of the actual test substance was examined in an Ames test (non-GLP, similar to OECD guideline 471). The material, dissolved in DMSO, was tested in 5 Salmonella typhimurium strains, TA 1535, TA 100, TA 1537, TA 1538, TA 98, in the presence and absence of a metabolic activation system at concentrations up to 5000 µg/plate. Results obtained by the incorporation method were confirmed by an independent pre-incubation experiment. Precipitation of the test item was observed from 2500 µg/plate onward. Mutagenicity was not detected. Due to the minor methodological deviation from OECD guideline 471 (only 2-Aminoanthracen was used as positive control with metabolic activation and the E. coli tester strain was missing), read across on a reliable gene mutation assay in bacteria with a structural analogue (CAS 386254-45-1) was performed.

 

An Ames test with the analogue substance (CAS 386254-45-1) was conducted according to OECD Guideline 471 and GLP using the S. typhimurium strains TA1535, TA100, TA1537, TA98 and the WP2 uvrA strain of E.coli. The substance was tested at a dose range of 22 to 5500 μg/plate in the standard plate test (SPT) and 4 – 2500 μg/plate in the preincubation test (PIT) in the presence and absence of mammalian metabolic activation (Aroclor-induced rat liver S-9 mix). In both tests (SPT and PIT) 3 test plates per dose and per control (vehicle and positive) were used. Precipitation of the test substance was found from about a test concentration of 500 μg/plate onward. A weak bacteriotoxic effect (slight decrease in the number of revertants and/or slight reduction in the titer) was occasionally observed in the standard plate test at about 5500 μg/plate and in the preincubation assay at doses ≥ 500 μg/plate. No increase in the number of his+ or trp+ revertants was observed in the standard plate test or in the preincubation test either with or without S-9 mix. According to the results of this study, the test item is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen.

 

An in vitro gene mutation study in mammalian cells was not conducted with the test substance itself, since adequate data from a structural analogue (CAS 106276-80-6) is available:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus, to test the potential of the analogue substance to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix) at dose levels of 3.2; 10; 31.6; 100; 316 and 1000 μg/mL. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions. Two independent experiments were performed. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) and diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability. The relative harmonised survival the relative total growth of the cells, the viability (colony-forming ability at the end of the 2 day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control throughout the study. Under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used. Under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

 

Furthermore, the cytogenic potential of the test item was not tested in mammalian cells. However, reliable, experimental data of a structural analogue (CAS 386254-45-1) is available.

The analogue substance was assessed in V79 cells in vitro for possible clastogenic or aneugenic activity leading to inducement of micronuclei both in the presence and in the absence of a metabolizing system at doses of 6.25 to 1500 μg/ml according to a proposal for a new OECD Guideline for the in vitro micronucleus test (1998) with the mixed population method (MP) and the mitotic shake off method (MSO). Test substance precipitation was observed macroscopically depending on the test conditions and the time of observation from about 200 μg/mI culture onward. 1000 cells were analyzed for micronuclei for each culture, i.e. 2000 cells for each test group. The negative controls (vehicle controls) gave frequencies within the range expected for the V79 cell line. Both of the positive control chemicals for clastogenicity, i.e. ethyl methane sulphonate and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei both in the MP and MSO. On the basis of the results of the study, the test substance did not cause any relevant increase in the number of cells containing micronuclei either without S-9 mix or after adding a metabolizing system. Under the experimental conditions of this assay, the test item is considered not to be a chromosome-damaging (clastogenic) agent nor does it induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells.

 

Based on the physico-chemical, structural as well as toxicological similarities of the two source substances and the target substance, the same outcome is assumed for all three endpoints for the actual substance as for the source substances.

 

in vivo:

A micronucleus test in mice is available for the actual test substance:

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, dissolved in water 0.5% CMC, was administered once orally to five males and 5 females at dose levels of 1000 mg/kg, 2000 mg/kg and 4000 mg/kg body weight. The animals were sacrificed and the bone marrow of the two femora was prepared 16, 24 and 48 houre after administration in the highest dose group of 4000 mg/kg body weight. In the test groups of 2000 mg/kg and 1000 mg/kg body weight, in the solvent control group and in the positive control group the 24-hour sacrifice intervals were investigated only. 1000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. An increase in the number of polychromatic erythrocytes containing micronuclei was not detected when compared to control. Clinical signs of toxicity, organ changes or an inhibition of erythropoiesis were not observed. Thus, it was concluded that the test substance did not induce cytogenetic damage in bone marrow cells.

 

As the study on the test substance itself is lacking the analytical verification for the test substance reaching the target tissue, the micronucleus test with an analogue substance (CAS 106276-80-6) is considered more reliable:

The analogue substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method (OECD guideline 474, GLP). For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. No relevant inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei. Under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect, nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

 

Based on the physico-chemical, structural as well as toxicological similarities of the two source substances and the target substance, the same outcome is assumed for all three endpoints for the actual substance as for the source substances.

 

In line with the negative in vivo results, the in vitro MNT with the analogue substance (CAS 386254-45-1) was negative, as described above.

Justification for classification or non-classification

Classification, Labelling, 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 mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.