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EC number: 283-392-8
CAS number: 84605-29-8
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.
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).
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.
Results on Genotoxicity Tests
Assay –(key study, present in section 7.6.1)
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
Cells (key study, present in section 7.6.1)
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.
shown in Table 2, in the absence of metabolic activation, the registered
material did not display mutagenic activity. In the presence of S9
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
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
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
difficulties with test solution preparation encountered and data not
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
Table 2: tk+/-Mouse
Lymphoma Assay Results
Tk+/-Mouse Lymphoma Assay
CAS 84605-29-8 / EC 283 -392 -8
Calcium Dialkyl dithiophosphate
transformation test (Supporting study, present in section 7.6.1)
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.
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.
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.
Transformation Test Results
*: expressed as
ratio between treated group vs. solvent control.
Micronucleus Test (in vivo) -(key
study, present in section 7.6.2)
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).
4: Mouse Micronucleus Test (in vivo)
CAS# / EC#
84605-29-8 / 283 -392 -8
Negative (doses:0, 7.13, 14.3 and 28.5 mg/kg)
Properties of the Registered Substance by Using QSAR Tool
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.
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.
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
is concluded that the registered substance is not expected to present a
significant risk for mutagenicity or carcinogenicity in humans.
et al. Mammalian Cell Mutagenesis: Maturation of Test Systems. Banbury
Report 2, 277-293, 1977
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.
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.
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.
R.H., and Plutnick, R.T. (1989). Carcinogenic potential of gasoline and
diesel engine oils.Fundamental and Applied Toxicology.13:545-553.
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
U., and Shubik, P. (1963). Studies on promoting action in skin
carcinogenesis.Natl. Cancer Inst. Monogr.10, 489-507.
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.
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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