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Test material CAS# 68909-93-3/EC# 272 -723 -1, Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts,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 included several endpoints to address the genotoxic potential of the test material: a) the ability to induce mutations in bacterial (, experimental data), b) the ability to induce mutations in mammalian cells (tk+/-mouse lymphoma assay, data obtained by read across); c) chromosome aberration (in vivomouse micronucleus assay, data obtained by read across). These studies are reliable (Klimitch code 1 for experimental data, 2 for read across data).

Negative results were obtained for EC 272-723-1 in theassay. By bridging to analogous substances, the test material was expected to be non-mutagenic (in vitro tk+/-mouse lymphoma assay) and non-crystogenic (in vivomouse micronucleus assay). 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 vitro bacteria gene mutation assay (AMES) has been conducted on CAS# 68909-93-3/EC# 272 -723 -1, 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







Mutagenicity Assay- in Mammalian Cells (key study, by read across to analogous substances, present in section 7.6.1)

Mutagenicity potential in mammalian cells was not evaluated for EC 272-723-1, however experimental data on structurally related substance EC 270-608-0 is available and suitable for read-across (justification for read across was shown in previously in this CSR).

In vitromammalian gene mutation potential at thymidine kinase (TK) locus was measured using L5178Y mouse lymphoma cell line after treated with EC 270-608-0, and negative response was observed with/without S-9 treatment. Based on this data, EC 272-723-1 was predicted to be non-mutagenic. 


Mouse Micronucleus Test (in vivo) -(key study, by read across to analogous substances, present in section 7.6.2)

 “Mammalian Erythrocyte Micronucleus Test” was available for two analogous substances. In both studies, 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. CAS# 68909-93-3 is therefore estimated to be non-clastogenic (Table 4).

Table 4: Mouse Micronucleus Test (in vivo)




CAS# 84605-29-8

EC# 283-392-8


Negative (doses:0, 7.13, 14.3 and 28.5 mg/kg)




Negative (read across)

CAS# 4259-15-8

EC# 224-235-5


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 prediction on DNA binding for both parental substance and 42 possible metabolites, and supported the conclusion that the test material is non-genotoxic.

Other Relevant Evidence:

No repeated dose toxicity studies were available for CAS 68909-93-3, but studies conducted on analogous substances suitable for read-across. For example, a 28-day repeated dose study via oral gavage, and a reproduction/development toxicity screening test are available for a CAS# 4259-15-8; an OECD 422 study was performed on CAS# 68457-79-4 (chapter 7.5 and 7.8). In these studies, the test substances were 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.


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


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:

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.