COAGULANT 122 (SOLID)

Endpoint summary

Additional information

Bacterial mutation assay:

In this in vitro assessment of the mutagenic potential of Coagulant 122 (solid), histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) were exposed to the test substance, diluted in water which was also used as a negative control.

Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

In the preliminary toxicity test with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.

No evidence of mutagenic activity was seen at any dose level of Coagulant 122 (solid) in either mutation test.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, when tested in water, Coagulant 122 (solid) was not mutagenic in this bacterial system.

Mammalian chromosome aberration test:

A study was performed to assess the ability of Coagulant 122 (solid) to induce chromosomal aberrations in human lymphocytes cultured in vitro.

Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treaunent time, cell division was arrested using colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of Coagulant 122 (solid) to cultured human lymphocytes, the mitotic index was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphæe analysis:

First test

Without S-9 mix, 18 hour harvest: 2500, 1250 and 625 µg/mL.

With S-9 mix, 18 hour harvest: 5000, 2500 and 1250 µg/mL.

Second test

Without S-9 mix, 18 hour harvest: 5000, 2500 and 1250 µg/mL.

With S-9 mix, 18 hour harvest: 5000, 2500 and 1250 µg/mL.

In the absence of S-9 mix, treatment with 2500 µg/mL in the first test and with 2500 and 5000 µg/mL of Coagulant 122 (solid) in the second test caused statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations.

ln the presence of S-9 mix, Coagulant I22 (solid) did not cause any statistically significant increases in aberrant cells.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells.

It is concluded that Coagulant 122 (solid) has shown evidence of clastogenic activity in the absence of S-9 mix but not in its presence, in this in vitro cytogenetic test system.

In vivo mammalian cell gene mutation test :

This study was designed to assess the potential induction of micro nuclei by Coagulant 122 (solid) in bone marrow cells of mice. Mice were treated with a single acute intraperitoneal administration of the test substance by injection at a dosage of 400, 800, 1600 mg/Kg. A preliminary toxicity test had previously shown 1600 mg/kg to be approximately the maximum tolerated dosage.

The test substance and negative control were administrated by intraperitoneal injection. The negative control group received the vehicle, purified water. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marow smears were obtained from five male and five female animals in the negative control and test substance groups at 2 sampling times; these being 24 or 48 hours after dosing. Bone marrow smears were obtained from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 1000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

Mice treated with Coagulant 122 (solid) did not show any significant increase in the frequency of micronucleated polychromatic erythrocytes at either sampling time.

There was no significant decrease in the ratio of polychromatic to normochoromatic erythrocytes after treatment of the animals with Coagulant 122 (solid).

The positive control compound, mitomycin C, produced large, highly significant increase in the frequency of micronucleated polychromatic erythrocytes together with decreases in the ratio of polychromatic to normochromatic erythrocytes.

It is concluded that Coagulant 122 (solid) has not shown any evidence of causing chromosome damage in this in vivo test.

Justification for selection of genetic toxicity endpoint
One study can not be selected as there are 2 in vitro and one in vivo key studies available.

Short description of key information:
Bacteria reverse mutation assay: perforemed according to OECD Guideline 471 in S. typhimurium TA 1535, TA 1537, TA 98, TA 100. Negative Ames (with and without metabolic activation)
In vitro mammalian chromosome aberration test: performed according to OECD Guidance 473 and EU Method B.10 in cultured human lymphocytes. Positive in vitro cytogenetics (with metabolic activation)
In vivo mammalian cell gene mutation test : performed according EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Negative in vivo bone marrow micronucleus

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The positive result in the in vitro cytogenetics assay gives some cause for concern with respect to mutagenicity. In accordance with the testing strategy, a bone marrow micronucleus test has been conducted. A negative result was obtained.

According to EC criteria, the notified substance need not be classified as germ cell mutagenis.