Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate)

Endpoint summary

Additional information

Test material CAS# 4259-15-8,Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate),isgenerically referred to as zinc dialkylthiophosphate (ZDDP). It is used in commerce as multi-functional anti-wear and anti-oxidation inhibitor performance components in passenger motor oils, diesel engine oils and industrial oils such as hydraulic lubricants.

The present dossier includes several testing approaches in attempt to measure the genotoxic potential of the test material: a) the ability to induce mutations in bacterial () or in mammalian cells (tk^+/-mouse lymphoma assay); b) chromosome aberration (in vivomouse micronucleus assay), c) cell transformation test using BALB/3T3 cell line. These studies are reliable without restriction (Klimitch code 1).

Negative results were obtained in theassay and thein vivomouse 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 treatment. 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)

In vitrobacteria gene mutation assay () has been conducted on this material, and 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).

Table 1:Assay Result

CAS#	Result
4259-15-8	Negative

 

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

In vitromammalian gene mutation potential at thymidine kinase (TK) locus was measured using L5178Y mouse lymphoma cell line after treated with various concentrations of the test material. A test substance was judged positive if there is a positive dose response and one or more of the three highest doses exhibit a mutant frequency which is 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 S9 microsomal enzyme, 3 independent tests were conducted on two samples of the test material: (study i) study disqualified due to contamination; (study ii) cultures treated with a series of concentrations produced total growth range from 3 to 44%, and 7/7 of the cultures exhibited positive response; (study iii) 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 , however, this finding was confounded by lack of reproducibility between repeat experiments, and the observed mutagenic activity concurrent with the presence of extensive cytotoxic damage at high doses. 

Study has shown that 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 (Mezayen,et al,2007).It is therefore postulated that the positive responses occurred at high ZDDP doses were partially due to cytotoxic concentrations, not direct effect(s) of metabolic transformation of test substance on mammalian DNA, or apparent genotoxicity was at least partially due to extragenomic damage(s).To support this hypothesis, the following substances were testedunder the same experimental conditions:a)zinc chloride,b)zinc oleate(technical difficulties with test solution preparation encountered and data not shown),c)calcium analogof a ZDDP (had previously shown positive activity in theseinvitromammalian 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 for mutagenicity. The results were consistent with previous studies which demonstrated zinc ion caused 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. Since zinc is not classified as carcinogen, the weight of evidence suggests that the test material is unlikely to be a mutagen.  

 

Table 2: tk^+/-Mouse Lymphoma Assay Results

 

CAS#	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 vitroBALB/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 lesionsin 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 supporting study in this dossier for the sake of completion.

As shown in Table 3, the test substance demonstrated transformation activity with S-9 activation, and statistically significant increases in transformation frequencies occurred only at the highest tested dose which 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 on such as cell proliferation, altered intercellular gap junction communication, ability to inhibit or induce apoptosis,etc., which are induced by spontaneous changes or exogenous factors (false positive for genetic outcome). 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 cell transformation.

Table 3:BALB/3T3 Transformation Test Results

CAS#	W/O S9	W/S9
4259-15-8	Negative	Positive
Calcium Dialkyl dithiophosphate	Not tested	Negative
ZnCl2	Not tested	Positive

 

  *: expressed as ratio between treated group vs. solvent control.

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, or at any harvest time point, or dose levels in mice (Table 4).

Table 4: Mouse Micronucleus Test (in vivo)

CAS#	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 by 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%~3% ZDDP, in rodent species yield limited number or no tumors in treated animals (Kaneet al,. 1984; McKee and Pryzygoda, 1987; Saffiotti and Shubik, 1963; McKee and Plutnick, 1989; Schreiner and Mackerer, 1982). Evidence supports 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. Mammalian Cell Mutagenesis: Maturation of Test Systems. Banbury Report 2, 277-293, 1977

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. Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin. (2007)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.), pp705-712. Battelle Press.

 

Short description of key information:
A SUMMARY OF CAS# 4259-15-8/EC# 224-235-5 GENOTOXICITY TEST RESULTS.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The weight of evidence suggests that the test substance is not expected to present a significant risk for mutagenicity or carcinogenicity in humans, therefore classification is not required in accordance with Directive 67/548/EEC and EU CLP (Regulation (EC) No. 1272/2008). Theories of justification present in the above “Discussion” section.