Registration Dossier

Administrative data

Key value for chemical safety assessment

Additional information

Test material CAS# 84605-29-8,Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl 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 includes several testing approaches in attempt to measure the genotoxic potential of the registered 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, positive results were observed in the tk+/-mouse lymphoma assay and 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 registered 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, and the frequencies of reverse mutations in bacteria were not significantly changed after exposure to various concentrations of the registered material, with/without S9 mixture (Table 1).

Table 1: AMES Assay Result

CAS#

Result

84605-29-8

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 registered 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, in the absence of metabolic activation, the registered material did not display mutagenic activity. In the presence of S9 microsomal enzyme, cultures treated with 3 highest doses of test material exhibited mutant frequency ranged from 16.3 to 2.0 times the mean mutant frequency of the solvent controls, the total growth of the cultures ranged from 1% to 72%; also, a correlation between cytotoxicity and mutation frequency was observed: as cell viability increased, the mutation frequency ratio (treated group/vehicle control) was dramatically decreased.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). Therefore, high incidence of cell death after ZDDP treatment may confound the outcome of the test and result in false positive.

To support the hypothesis that the observed positive responses were at least partially due to a cytotoxic concentration, not direct effect(s) of metabolic transformation of test substance on mammalian DNA, 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 analog of a ZDDP (had previously shown activity in these invitromammalian cell assays). The following results were obtained:a): Zinc chloride showed high degree of cytotoxicity and positive for mutagenicity. The results were consistent with a previous work which demonstrated zinc ion caused cytotoxicity and mutagenicity in similarly cultured mammalian cell systems (Amaker et al., 1979);b)calcium dialkyl dithiophosphate did not show mutagenicity. Taken together, the data suggest the dialkyldithiophosphate portion of ZDDP molecule is non-mutagenic, but the zinc may have been the causative agent under the test conditions. Since zinc is not classified as carcinogen, the weight of evidence suggests that the registered ZDDP material is unlikely to be a mutagen.  

Table 2: tk+/-Mouse Lymphoma Assay Results

 

 

Test Sample

Tk+/-Mouse Lymphoma Assay

W/O S9

W/S9

CAS 84605-29-8 / EC 283 -392 -8

Negative

Positive

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 endpoint is not required 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 registered substance demonstrated transformation activity with/without S-9 activation, and statistically significant increases in transformation frequencies occurred at high doses 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 tests is 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 exogenous factors or spontaneous changes. So this particular assay is sensitive to these epigenetic changes, and predisposes to giving false positive conclusion for genetic outcome. As complementary to the tk+/-Mouse Lymphoma Assay, the transformation studies on this ZDDP substance demonstrated the zinc subcomponent, not the dialkyldithiophosphate portion, may have been the causative agent for epigenetic changes if there was any.

 

Table 3:BALB/3T3 Transformation Test Results

Test Sample

W/O S9

W/S9

CAS 84605-29-8 / EC 283 -392 -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# / EC#

Result

84605-29-8 / 283 -392 -8

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

 

 

Intrinsic Properties of the Registered 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 7 possible metabolites, and supported the conclusion that the test material is non-genotoxic.

Other Relevant Evidence:

Available repeated dose studies performed on structural analogues, such as 28 day repeated dose oral study and an OECD 421 reproduction /development toxicity screening test performed on CAS# 4259-15-8, or an OECD 422 study performed on CAS# 68457-79-4, were included in this registration dossier (chapter 7.5 and 7.8). These ZDDP substances were unable to induce hyperplasia or pre-neoplastic lesions in the test animals.

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 registered 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# 84605-29-8/EC# 283-392-8 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.