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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Additional information

EC 234-277-6 is generically referred to as zinc dialkylthiophosphate (ZDDP), there are genotoxic data available for this substance. However, sufficient data is available for other structure related ZDDP materials. All the ZDDP materials are produced under similar manufacturing procedures and 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. They share great chemical similarity and have been considered as a category (HPV Test Plan).

Table 1. Substance identities from ZDDP category

CAS#

EC#

Substance Name

84605-29-8

283-392-8

Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts

68457-79-4

270-608-0

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

68909-93-3

272-723-1

Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-Pr) esters, zinc salts

4259-15-8

224-235-5

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

 

Studies of Genotoxicity and Related Endpoints

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

In vitro bacteria gene mutation assay (AMES) has been conducted, and the frequencies of reverse mutations in bacteria were not significantly changed after exposure to various concentrations of the ZDDP materials, with/without S9 mixture (Table 2). The study is reliable without restriction (Klimitch code 1).

Table 2: AMES 

CAS#

Result

84605-29-8

Negative

68909-93-3

Negative

4359-15-8

Negative

 

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

In vitro mammalian 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 mean mutant frequency ratio between treated group and solvent control greater than 2.

As shown in Table 3, the registered material did not display mutagenic activity in the absence of metabolic activation. Mutagenic activity after treatment in S9 microsomal enzyme preparations was observed, but only at high dose levels which associated with high degree of cytotoxicity. As cell viability increased, the mutation frequency ratiotreated group/vehicle controlwas dramatically decreased.High incidence of cell death after ZDDP treatment may confound the outcome of the test and result in false positive. 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).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, 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 invitro mammalian cell assays). 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).However, 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 3: tk+/-Mouse Lymphoma Assay Results

 

CAS#

Tk+/-Mouse Lymphoma Assay

W/O S9

W/S9

84605-29-8

 

Negative

Positive

Dose (ul/ml)   Total growth         Ratio

0.031                   1%                   16.3

0.018                  72%                   2.0

68457-79-4

 

Negative

Negative

Dose (ul/ml)   Total growth         Ratio

0.018                   77%                   1.0

0.013                  91%                    0.9

4259-15-8

 

Negative

Equivocal:because dose response is observed even though the ratio < 2.

 

Dose (ul/ml)   Total growth           Ratio

0.021                 27                      1.9

0.016                 61                      1.8

0.012                 77                      1.2

0.0089               77                       1.2

0.0067               62                      1.0

0.005                 91                      1.0

0.0038               96                      1.3

0.0028               90                      1.1

0.0021               89                      1.0

0.0016               91                      1.0

Calcium Dialkyl dithiophosphate

not tested

Negative

ZnCl2

not tested

Positive

 

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

In theMammalian 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

84605-29-8

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

4259-15-8

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

 

Intrinsic Properties of the Registered Substance by Using QSAR Tool

QSAR analysis of the registered material and possible metabolites show lacking any structural alerts for DNA binding (OECD toolbox 1.1.01), and is predicted to be a non-genotoxic.

Other Relevant Evidence:

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.

 
Short description of key information:
EC 234-277-6 is unlikely to be genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In accordance with EU CLP (Regulation (EC) No. 1272/2008) classification is not required for genotoxicity.