Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts

Endpoint summary

Additional information

Test material EC 270-608-0, Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts, is generically 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 (by read across to analogous substance) 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 (Klimitch code 1 for experimental data, 2 for read across data).

Negative result was estimated forassay; negative results were obtained in tk^+/-mouse lymphoma assay andin vivomouse micronucleus assays. However, positive results were observed in the BALB/3T3 transformation test 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 isNOTgenotoxic.

 

Study Results on Genotoxicity Tests

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

In vitro bacteria gene mutation assay (AMES) has been conducted on analogous substances (EC 283-392-8 and EC 272-723-1) suitable for read-across. The frequencies of reverse mutations in bacteria were not significantly changed after exposure to various concentrations of the test materials, with/without S9 mixture. By read across (detailed justifications present in the robust summary), CAS#68457-79-4 is estimated to be non-mutagenic in bacteria(Table 1).

Table 1: AMES Assay Results

CAS# / EC#	Average MW	Result
84605-29-8 / 283 -392 -8	576	Negative
68457-79-4 / 270 -608 -0	576	Negative (read across)
68909-93-3 / 272 -723 -1	632	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 materials. 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, the test material did not cause cytotoxicity or mutagenic activity in the absence/presence of metabolic activation at any dose groups. 

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

 

CAS# / EC#	tk^+/-Mouse Lymphoma Assay
	W/O S9	W/S9
68457 -79 -4 270 -608 -0	Negative	Negative

 

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 without S-9 activation, but did not achieve statistical significance. Also, great extent of cell loss was observed at various dose levels. Published study has shown that stressed/ injured /necrotic cells release various molecules that can trigger biological responses (an indirect effect from treatment with the test substances) in the remaining viable cells (Mezayen,et al,2007). 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).It is therefore postulated that the positive responses occurred at high doses were likely secondary to cytotoxicity or apparent genotoxicity that is actually due to extragenomic damage(s).

To support the hypothesis that the observed positive responses were due to a cytotoxic concentration, not direct effect(s) of metabolic transformation of test substance on mammalian DNA, two substanceswere tested under the same conditions:zinc chloride, andcalcium analog of a ZDDP (had previously shown activity in these invitromammalian cell assays). Zinc chloride showed high degree of cytotoxicity and transformation activity; but these effects were not found in calcium dialkyl dithiophosphate treated groups. Taken together, the data suggest the dialkyldithiophosphate portion of ZDDP molecule lacks transformation activity, whereas the zinc portion may have been the causative agent under the test conditions. Since zinc is not classified as genotoxic or carcinogenetic, the weight of evidence suggests that the tested material is unlikely to be genotoxic or exert its effect via epigenetic mechanisms. 

 

Table 3:BALB/3T3 Transformation Test Results

Test Sample	W/O S9	W/S9
68457-79-4 / 270 -608 -0	Positive No statistical significance was achieved, positive because transformation activity was observed.	Not tested
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)

“Mammalian Erythrocyte Micronucleus Test” is not available for the test material, but has been conducted on analogous substances (EC 283-392-8 and EC 272-723-1) suitable for read-across. In these tests, 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). By read across (detailed justifications present in the study report robust summary), CAS#68457-79-4 is estimated to be non-clastogenic.

Table 4: Mouse Micronucleus Test (in vivo)

CAS# / EC#	Average MW	Result
84605-29-8 / 283 -392 -8 (analogue substance, data source)	576	Negative
68457-79-4 / 270 -608 -0 (target)	576	Negative (read across)
68909-93-3 / 272 -723 -1 (analogue substance, data source)	632	Negative

 

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 11 possible metabolites, and supported the conclusion that the test material is non-genotoxic.

Other Relevant Evidence:

Available repeated dose studies were performed on this material (OECD 422), or structural analogue (such as 28 day repeated dose oral study, and an OECD 421 reproduction /development toxicity screening test performed on CAS# 4259-15-8). 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# 68457-79-4/EC# 270-608-0 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.