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Additional information

Reliable, relevant and adequate data on the genetic toxicity of AAPBs is available from several in vitro gene mutation studies in bacteria (Ames Tests), one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) and onein vivocytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test). All tests were consistently negative. There is no evidence for a genotoxic potential of AAPBs.

Relevant data in detail

In vitrodata

In the keyin vitroreverse gene mutation assay in bacteria performedaccording to EU Method B.14 (Version Commission Directive 92/69/EEC), strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 of Salmonella typhimurium were exposed to C8-18 and C18 unsatd. AAPB. Test was performed with concentrations up to and including cytotoxic concentrations in the absence and the presence of mammalian metabolic activation.

No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including its cytotoxic limit. The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.

The adopted OECD TG 471 (1997) requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, AAPB is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 and EU Method B.13/14 (Version Commission Directive 92/69/EEC) without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of AAPB in this bacterial test system.

Consistently negative results were also seen in several further supporting in vitro gene mutation assays in bacteria performed on Coco AAPB.

In a mammalian cell gene mutation assay according to OECD Guideline 476 (1997) and EU Method B.17 (2008), the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y was tested.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix: 2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL

with S9 mix: 4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix: 10; 20; 40; 50; and 60 µg/mL

with S9 mix: 40: 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % relative total growth was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups. In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 106cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 106cells. The solvent controls remained within the range of the historical data. Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

In vivo data

The cytogenetic activity of the AAPBs was tested in a OF1 (I. O. P. S. Caw) mouse bone marrow micronucleus assay, performed as described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975). 5 male and 5 female animals were treated i. p. with Coco AAPB (30 % a. i). The test method by W. Schmidt is almost equivalent to the procedure described by OECD guideline 474. In a preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of ≥ 100 mg/kg bw/day. At doses ≥ 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

The mean number of micronucleated erythrocytes/1000 polychromatic erythrocytes in males and female mice at 20 and 200 mg/kg bw/day were unaffected compared to the negative controls. The administration of 100 mg cyclophosphamide/kg bw serving as the positive control led to clearly elevated numbers of micronucleated erythrocytes.

It can be concluded, that Coco AAPB (30 % a. i) induced no clastogenic effect in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

Conclusion

Reliable, relevant and adequate data on the genetic toxicity of AAPBs is available from several in vitro gene mutation studies in bacteria (Ames Tests), onein vitrogene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) and one in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test). The full set of mutagenicity tests required by REACH Regulation Annexes VII and VIII is covered with the studies, whereas the information requirement on an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study is fulfilled in accordance to Annex VIII, column 2 by adequate data from an in vivo cytogenicity study. The studies were performed on Coco AAPB or C8 -18 AAPB.

There was no evidence of mutagenic or clastogenic intrinsic properties in any of the performed studies.

The genotoxic potential of the whole group of AAPBs is assumed to be similar. As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the AAPBs. Thus, the use of studies performed on individual members of this group of substances as read-across for the whole group is justified without restrictions.

In conclusion there is no evidence for a genotoxic property for AAPB.


Short description of key information:
In vitro data from several gene mutation studies in bacteria (Ames Tests) on Coco AAPB and one study on C8 -18 and C18 unsatd. AAPB, one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) performed on C8-18 AAPB and one cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test) on Coco AAPB are available. All tests were consistently negative. There is no evidence for a genotoxic potential of AAPBs.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

AAPBs considered to have no genotoxic properties as shown in several gene mutation studies in bacteria (Ames Tests), one in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) and one in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test).

Therefore, AAPBs do not comply with the classification requirements regarding germ cell mutagenicity outlined in regulation (EC) 1272/2008 or the former European directive on classification and labelling 67/548/EEC.