Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

The genotoxicity assessment of CASRN 54261-67-5 is based on read across from test data with an analog substance (CASRN 4529-15-8). This is a conservative approach to evaluating the potential genotoxicity of CASRN 54261-67-5, which is a larger molecule. Therefore, CASRN 54261-67-5 is expected to have a lower potential for bioavailability and toxicity than the analog substance. CASRN 54261-67-5 also contains tetrapropenyl phenol (also known as Phenol, dodecyl-, branched; EC Number 310-154-4)as an impurity. Tetrapropenyl phenol is not mutagenic or genotoxic as demonstrated by the test data for this substance, which can be found in the registration dossier for this material

The analog test material zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CASRN 4259-15-8) is generically referred to as zinc dialkylthiophosphate (ZDDP). It is used in commerce as an anti-wear, anti-oxidation, and corrosion inhibitor additive applied to passenger motor, diesel engine, and industrial oils.

In attempt to assess the genotoxic potential of the analog test material, the present dossier includes several testing approaches: a) the ability to induce mutations in bacterial cells and mammalian cells (TK+/- mouse lymphoma assay); b) chromosome aberration (in vivo mouse micronucleus assay), and c) cell transformation test using BALB/3T3 cell line. These studies are reliable without restriction (Klimisch code 1). 

Negative results were obtained from in vivo mouse micronucleus assays. However, equivocal results in the TK+/- mouse lymphoma assay and positive results in BALB/3T3 transformation test were observed after rat liver S9 microsomal enzyme treatments. All the assays were thoroughly reviewed and assessed in accordance to REACH and OECD guidance. The following sections cover a spectrum of evidence/justifications and the weight of evidence suggests that the test material is not genotoxic.

 

Study Results on Genotoxicity Tests

Mutagenicity Assay – (key study, present in section 7.6.1)

An in vitro bacteria gene mutation assay has been conducted on this material. The frequencies of reverse mutations in bacteria were not significantly changed after exposure to various concentrations of the test material, with/without S9 mixture (Table 1).

 

Assay Result

CASRN

Result

4259-15-8

Negative

 

Mutagenicity Assay- In Mammalian Cells (key study, present in section 7.6.1)

In vitro mammalian gene mutation potential at the thymidine kinase (TK) locus was measured using L5178Y mouse lymphoma cell line after treatment with various concentrations of the test material. A test substance was judged positive if there was a positive dose response and one or more of the three highest doses exhibit a mutant frequency which was two-fold greater than the background level.

As shown in Table 2, in the absence of metabolic activation, the test material did not display mutagenic activity. In the presence of the S9 microsomal enzyme, four independent tests were conducted on two samples of the test material:(study i)the results were found to be equivocal; (study ii) study was disqualified due to contamination; (study iii) cultures treated with a series of concentrations produced total growth range from 3 to 44% and 7/7 of the cultures exhibited positive response; and (study iv) cultures treated with a series of concentrations produced total growth range from 27 to 96% and 0/7 of the cultures exhibited mutant frequencies which were significantly greater than the mean frequency of the solvent controls. However, a dose-dependent response was noted. The test material was eventually determined to be equivocal for mutagenicity. In addition, these findings were confounded by the lack of reproducibility and the presence of extensive cytotoxic damage at high doses. 

A study has shown how stressed/injured/necrotic cells release various molecules that can trigger biological responses in the remaining viable cells via indirect effect(s) after treatment with test substances (Mezayenet al.,2007). It is therefore postulated that the positive responses occurred at high ZDDP doses were partially due to cytotoxic concentrations, and not as direct effect(s) of metabolic transformation of test substance on mammalian DNA or apparent genotoxicity (i.e., extragenomic damages).

To support this hypothesis, the following substances were tested under the same experimental conditions:a)zinc chloride,b)zinc oleate (technical difficulties with test solution preparation was encountered and data was not shown),c)calcium analog of a ZDDP (which previously had shown positive activities inin vitromammalian cell assays). And the following results were obtained:a)zinc chloride showed high degree of cytotoxicity (the total growth ranged from 2% to 61%) and positive results for mutagenicity. The results were consistent with previous studies which demonstrated that the zinc ion caused the cytotoxicity and mutagenicity in similarly cultured mammalian cell systems (Amakeret al., 1979);b)calcium dialkyl dithiophosphate did not exhibit mutagenicity and relative higher cell viability was obtained (the total growth ranged from 17% to 74%). Taken together, the data suggest the dialkyldithiophosphate portion of the ZDDP molecule is non-mutagenic. The zinc subcomponent may have been the causative agent under the test conditions. However, Since zinc is not classified as carcinogen, the weight of evidence suggests that the test material is unlikely to be a mutagen.

 

TK+/- Mouse Lymphoma Assay Results 

 

CASRN

                        Tk+/-Mouse Lymphoma Assay

 W/O S9

 W/S9

4259-15-8

 

Test sample 1

Negative

Test sample 1

Equivocal

Test sample 1

Positive

Found contamination and erratic dose-response relationship in toxicity

Test sample 1

Positive

The treated group showed significantly greater mean mutant frequency than the level of the solvent controls.

Test sample 2

Equivocal

The treated group did NOT show significant increase in mean mutant frequency comparing to the level of the solvent controls, but a dose response was observed.

Calcium Dialkyl dithiophosphate

Not tested

Negative

ZnCl2

Not tested

Positive

 

 

BALB/3T3 Transformation Test- (Supporting study, present in section 7.6.1)

In vitro BALB/3T3 transformation test protocol (1982) was designed to assess the ability of chemicals to induce changes in the morphological and growth properties of cultured mammalian cells. The observed changes were presumed to be similar to phenotypic changes that accompany the development of neoplastic or pre-neoplastic lesions in vivo. The test procedures were different from the two-stage protocols described in the OECD Series on Testing and Assessment No. 31 (2007). Considering this is not a required endpoint for REACH registration, the BALB/3T3 transformation test was regarded as a supporting study in this dossier.

As shown in Table 3, the test substance demonstrated transformation activity with S-9 activation. Statistically significant increases in transformation frequencies occurred only at the highest tested dose which was associated with noticeable cytotoxicity. Similar to the strategy used in the TK+/-Mouse Lymphoma Assay, calcium dialkyldithiophosphate and ZnCl2were tested for transformation activity. Negative and positive results were observed, respectively.

BALB/3T3 transformation test is sensitive to epigenetic changes and widely used for mechanistic studies such as cell proliferation, altered intercellular gap junction communication, and the ability to inhibit or induce apoptosis, which are induced by spontaneous changes or exogenous factors (or false positives). As complementary to the tk+/-Mouse Lymphoma Assay (genetic events), the transformation studies on this ZDDP substance demonstrated the zinc subcomponent, not the dialkyldithiophosphate portion, may have been the causative agent for epigenetic events which ultimately led to the cell transformation.

 

BALB/3T3 Transformation Test Results

CASRN

W/O S9

W/S9

4259-15-8

Negative

Positive

Calcium Dialkyl dithiophosphate

Not tested

Negative

ZnCl2

Not tested

Positive

 

Mouse Micronucleus Test (in vivo) (key study, present in section 7.6.2)

In the Mammalian Erythrocyte Micronucleus Test, no statistically significant increases in micronucleated polychromatic erythrocytes over the levels observed in the vehicle controls were observed in either sex, at any harvest time point, or at any dose levels of the test material applied to mice (Table 4).

 

Mouse Micronucleus Test (in vivo)

CASRN

Result

4259-15-8

Negative (doses: 0, 6, 12 and 24 mg/kg)

  

Intrinsic Properties of the Test Substance by Using QSAR Tool

The test material was profiled with DNA binding and Benigni/ Bossa rulebase grouping methods using OECD toolbox 1.1.01. QSAR analyses showed negative predictions on DNA binding potentials for parental and 45 possible metabolites and supported the conclusion that the test material is non-genotoxic.

 

Other Relevant Evidence

A 28-day repeated dose study via oral gavage and a reproduction/development toxicity screening test are available for this substance. In these studies, the test substance was unable to induce hyperplasia and/or pre-neoplastic lesions.

Published carcinogenicity studies using fresh motor oil, commonly containing 1% to 3% ZDDP, and rodents yielded a limited number of or no tumors in treated animals (Kaneet al,. 1984; McKee and Pryzygoda, 1987; Saffiotti and Shubik, 1963; McKee and Plutnick, 1989; and Schreiner and Mackerer, 1982). Evidence from these studies supports the premise that ZDDP materials lack carcinogenic potential.

 

CONCLUSION

It is concluded that the test substance is not expected to present a significant risk for mutagenicity or carcinogenicity in humans.

 

Reference:

Amacher et al. (1977) Mammalian Cell Mutagenesis: Maturation of Test Systems. Banbury Report 2, 277-293.

 

Kane, M., LaDov, E., Holdworth, C., and Weaver, N. (1984). Toxicological characteristics of refinery streams used to manufacture lubricating oils.Amer. J. Ind. Med.5:183-200.  

 

Mezayen, R.EI., Gazzar, M.EI., Seeds, M.C., McCall, C.E. and Nicolls, M.R. (2007) Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin.Immunology Letters.111:36-64.

 

McKee, R.H., and Przygoda, R. (1987). The genotoxic and carcinogenic potential of engine oils and highly refined lubricating oil.Environ. mutagen.9(suppl. 8), 72 Abstract.

 

McKee, R.H., and Plutnick, R.T. (1989). Carcinogenic potential of gasoline and diesel engine oils.Fundamental and Applied Toxicology.13:545-553.

 

Renznikoff,, Bertram, J.S., Brankow, D.S. and Heidelberger, C. (1973). Quantitative and qualitative studies of chemical transformation of cloned C3H mouse embryo cells sensitive to post-confluence inhibition of cell division.Cancer Res.33:3239-3249.

 

Saffiotti, U., and Shubik, P. (1963). Studies on promoting action in skin carcinogenesis.Natl. Cancer Inst. Monogr.10, 489-507.

 

Schreiner,, and MacKerer, C.R., (1981). Mutagenic Testing of Gasoline Engine Oils.InPolynuclear Aromatic Hydrocarbons:Chemical and Biological Effects(M. Cooke, A.J. Dennis, and G.L. Fisher, Eds.), pp 705-712. Battelle Press.


Short description of key information:
Testing for genotoxicity of CAS# 543261-67-5 is waived based on available information for lower molecular weight zinc alkyl dithiophosphate substances. A summary of the assessment of genotoxicity for the highest molecular weight alkyl dithiophosphate evaluated for REACH registration, CAS# 4259-15-8/EC# 224-235-5, is provided.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The weight of evidence review for the test material, zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate), (CASRN 4529-15-8) indicates the substance is not expected to present a significant risk for mutagenicity or carcinogenicity in humans; and therefore classification is not required in accordance with Directive 67/548/EEC and EU CLP (Regulation (EC) No. 1272/2008). Read across from CASRN 4529-15-8 is a conservative approach to evaluating the potential genotoxicity of CASRN 54261-67-5, which is a larger molecule; and therefore, CASRN 54261-67-5 is expected to have a lower potential for bioavailability and toxicity.