Blue MOP 6314

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The testing strategy about this substance are made following the ECHA guidance R.7a R.7.7 -1 Flow chart of the mutagenicity testing strategy.

1) Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. modified according to Prival and Mitchell.

CONCLUSION:

Based on the results of this study it is concluded that the substance is mutagenic in the Salmonella ryphimurium reverse mutation assay and that it is not mutagenic in the Escherichia coli reverse mutation assay.

After the conclusion reported above, a REACH annex VIII criteria must be satisfied; therefore an in vitro mammalian cell gene mutation test has been conducted on test item.

2) In vitro Mammalian cell gene mutation test with L5178y mouse lymphoma cells (with indipendent repeat).

CONCLUSION:

This report describes the effects of test item on the induction of forward mutations atthe thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. The test was performed in three independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). A range finding study was performed to set dose levels for the subsequent mutation studies and

to establish the solubility of BLUE MOP 6314. Since it was poorly soluble in aqueous solution, the highest tested concentration was 900µg/ml exposure medium.

In the first experiment, the substance was tested up to concentrations of 160 and 800µg/ml in the absence and presence of 8 % (v/v) S9-mix, respectively. Incubation time was 3 hours. The substance was tested up to cytotoxic levels of 99 and 90% in the absence and presence

of S9-mix respectively. In the second experiment, BLUE MOP 6314 was tested up to concentrations of 450 and 800µg/ml in the absence and presence of 12 % (v/v) S9-mix, respectively. Incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9 metabolic activation

respectively. The substance was tested up to cytotoxic levels of 63 and 70% in the absence and presence of S9-mix respectively.

In the third experiment, the substance was tested up to concentrations of 425 and 900µg/ml in the absence and presence of 12 % (v/v) S9-mix, respectively. Incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9 metabolic activation

respectively. The substance was tested up to cytotoxic levels of 94 and 65% in the absence and presence of S9-mix respectively.

Mutant frequency at TK-locus Mutant frequencies in cultures treated with positive control chemicals were increased by 6-, 20- and 28-fold for EMS and by 9-, 16- and 13-fold for DMN. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. The substance did not induce a significant increase in the mutant frequency at the TK locus in the absence of S9 metabolic activation in the first experiment after a 3 hours treatment period.However after extension of the treatment period to 24 hours in the second experiment, test item induced 2.3- and 2.6-fold increases in the mutant frequency at the TK locus at concentrations of 300 and 350µg/ml respectively. Verification of this result was performed in an additional third experiment, in which the substance induced an up to 51-fold increase at the concentration of 300µg/ml and test item induced 2.4- to 3.6-fold increases in the mutant frequency at the TK locus at concentrations of 350, 400 and 425µg/ml.

The mutagenic effect observed in the second experiment was observed at intermediate concentrations (300 and 350µg/ml), moderate toxicity (Relative Total Growth of 62 and 33%) and the mutagenic effect was not three-fold, although the increases were outside our historical control data range. The mutagenic effect observed in the third experiment was extremely high at one dose level (the mid-concentration of 300µg/ml). This extreme increase was observed only at one very toxic concentration (RTG = 9%). The increases observed at the three higher dose levels of 350, 400 and 425 were at moderate to severe toxicity and at two dose levels (350 and 425µg/ml) more than three-fold. Taken together, these increases are considered to be biologically relevant and the substance is considered to be mutagenic in the absence of S9-MIX.

In the presence of 8% (v/v) S9-mix induced no significant increase in the mutant frequency in the first experiment. This result was confirmed in two repeat experiments with modifications in the composition of the S9 concentration (12% (v/v)). Although in the presence of 12% (v/v) S9-mix the maximal toxicity was below the optimal 80%, this was considered acceptable, as the level of toxicity was still marked (70 and 65%) and in the presence of 8% (v/v) S9-mix showed sufficient toxicity (90%). Since was not soluble in the exposure medium up to and including a concentration of 8 mg/ml and also poorly soluble in dimethyl sulfoxide, the highest concentration that could be achieved was 900µg/ml.

In conclusion, the substance is mutagenic in the TK mutation test system in the absence of a metabolic activation system. The test item is not mutagenic in the TK mutation test system in the presence of a metabolic activation system under the experimental conditions described in this report.

Based on the results of this study it is concluded that the substance is mutagenic in the TK mutation test system in the absence of a metabolic activation system.

After the conclusion reported above, a REACH annex IX criteria must be satisfied; therefore further investigations with an in vivo test must be satisfied.

3) OECD Guideline 474 (in vivo Mammalian Erythrocyte Micronucleus Test)

The substance was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow.

Six groups each comprising 5 males and 5 females, received a single intraperitoneal injection.

Two groups were dosed with 100 mg/kg body weight, one group was dosed with 50 mg/kg body weight and one group was dosed with 25 mg/kg body weight. After dosing the animals of all the dose levels showed the following toxic signs: lethargy and a hunched posture. The animals of the dose levels of 100 and 50 mg/kg body weight also showed a rough coat and one of the animals dosed with 100 mg/kg body weight showed ataxia.

A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Bone marrow of the groups treated with test item was sampled 24 or 48 hours after dosing. Bone marrow from the negative control group was harvested at 24 hours after dosing only and bone marrow from the positive control group was harvested at 48 hours after dosing only.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes in both sexes. No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with test item. The animals of the groups treated with 100 mg/kg body weight with test item (males at the 24 and 48 hours sampling time and females at the 48 hours sampling time) showed a slight but variable decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, indicating that the substance is toxic with respect to the erythropoiesis.

The groups that were treated with cyclophosphamide showed a clear decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. It is concluded that the substance is not mutagenic in the micronucleus test under the experimental conditions described in this report.

4)OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Evaluation of DNA repair inducing ability of test item in male rat hepatocytes (in vivo rat hepatocyte DNA repair assay).

This report describes the DNA repair inducing ability of the substance in male Wistar rat hepatocytes, measured as unscheduled DNA synthesis (UDS).

The study procedures were based on the following guidelines:

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Guideline No. 486: Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo (adopted 21st July 1997).

- European Economic Community (EEC) Directive 2000/32lEC, Part B: Methods for the Determination of Toxicity; B.39: "UnscheduIed DNA synthesis (UDS) test with mammalian liver cells in vivo".

- Butterworth B.E. et al (1987) A protocol and guide for the in vivo rat hepatocyte DNA-repair assay. Mutation Research, 189, 123-133.

Batch 37 of test item was a dark blue powder with a purity of 95.2%. The test substance was suspended in corn oil in the dose range finding study. However, since the substance suspended in corn oil was difficult to administer to the animals, in the main study the substance was dissolved or suspended in Milli-Ro water.

Dose groups received a single oral dose of the substance. Hepatocytes were sampled 2-4 or 12-16 hours after dosing with 1000 and 2000 mg BLUE MOP 6314/kg body weight (b.w). No abnormalities were observed in both dose groups, with the exception of one animal of the highest dose group which showed the following toxic signs: lethargy, ataxia, rough coat and gasping for breath. The animals of the long treatment period had dark blue coloured faeces. Two slides per animal and three animals per data point were examined.

Corresponding vehicle treated groups served as negative controls (vehicle was Milli-Ro water). Hepatocytes of positive control animals treated with single oral doses of dimethylnitrosamine (DMN, 10 mg/kg b.w.) or 2-acetylaminofiuorene (2-AAF, 50 mg/kg b.w.) were harvested 2-4 or 12-16 hours after dosing, respectively. Two slides per animal and one animal for the control groups were examined.

As a result of oral dosing with the substance the net nuclear grain count (NNG) per coverslip and per animal, as well as the group average revealed no positive response in this assay. The percentage of cells in repair (repair taken as NNG 3 5), both per individual animal and for the group average, revealed no significant increase at any dose.

The results of the negative and positive controls were within the expected range. Therefore, it can be concluded that the test system was functioning correctly.

It is concluded that the substance is not genotoxic in the DNA-repair assay using hepatocytes obtained from male rat liver following in vivo exposure for 2-4 or 12-16 hours to the test substance up to concentrations of 2000 mg/kg b.w. under the conditions described in this report.

CONCLUSION:

Based on ECHA guidance R.7a R.7.7 -1 Flow chart of the mutagenicity testing strategy; after the negative result of two indipendent in vivo test (OECD 474 and OECD 486), the substance is considered not genotoxic and no further testing are required.

Justification for classification or non-classification

According to the CLP Regulation (EC n. 1272/2008) a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms.

After all investigations it can affirm that the test substance is not capable to induce permanent mutation inside the genetic structure, therefore according to the ECHA guidance R.7a R.7.7 -1 Flow chart of the mutagenicity testing strategy, no further testing at this level are necessary and no classification is warranted according to the CLP Regulation (EC 1272/2008).