Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 821-762-8 | CAS number: 4563-56-8
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Ames test: negative in TA98, TA100, TA1535 and TA1537 with and without metabolic activation
RA from CAS 4259-15-8
Mouse lymphoma assay: negative in L5178Y mouse lymphoma cells without metabolic activation, ambiguous in L5178Y mouse lymphoma cells with metabolic activation
RA from CAS 4259-15-8
BALB/3T3 Transformation Test: negative without metabolic activation and positive with metabolic activation
RA from CAS 4259-15-8
(Q)SAR prediction: no alerts found for genotoxicity using Vega v1.1.3 and OECD QSAR Toolbox v3.4
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- (strain with AT base pair at the primary reversion site is missing)
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon (hisG46, hisC3076, hisD3052); Lipopolysaccharide barrier (LPA); DNA excision repair (uvrB)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- mammalian microsomal enzymes
- Test concentrations with justification for top dose:
- with S9 mix; 25, 50, 100, 250, 1000, and 5000 µg/plate
without S9 mix: 1, 5, 10, 25, 100, 500 µg/plate
Confirmatory assay:
with S9 mix; 50, 100, 250, 500, 1000, and 5000 µg/plate
without S9 mix: 5, 10, 25, 50, 100, 500 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: multiple postive controls (depending on strain and metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 hours
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition - Evaluation criteria:
- TA98, TA100, WP2uvrA: A positive result must produce at least a 2-fold increase of the mean revertants per plate of at least one tester strain over the mean revertants per plate of the control. A dose response in the mean number of revertants per plate must also occur.
TA1535 and TA1537: A positive result must produce at least a 3-fold increase of the mean revertants per plate of at least one tester strain over the mean revertants per plate of the control. A dose response in the mean number of revertants per plate must also occur. - Key result
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- The test substance did not show mutagenic properties in TA98, TA100, TA1535 and TA1537 with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- January 1983 - October 1983
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- not specified
- GLP compliance:
- no
- Type of assay:
- other: mammalian cell gene mutation assay
- Target gene:
- The thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 using male Sprague-Dawley rats injected i.p. with 2:1 mixture of Aroclor 1242: Aroclor 1254 (in corn oil/200 mg/mL). 5 days post injection rats were decapitated and livers excised.
- Test concentrations with justification for top dose:
- (without S9): 0.0089, 0.0067, 0.0050, 0.0038, 0.0028, 0.0021, 0.0016, 0.0012, 0.00089 or 0.00067 µL/mL
(with S9): 0.021, 0.016, 0.012, 0.0089, 0.0067, 0.0050, 0.0038, 0.0028, 0.0021 or 0.0016 µL/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in suspension
DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h
- Expression time (cells in growth medium): 24 and 48 h (viability)
- Selection time (if incubation with a selection agent): 10-12 days.
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable.
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable.
STAIN (for cytogenetic assays): Not applicable.
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: 200 cells/plate
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy: No.
- Determination of endoreplication: No.
OTHER: Calculation of Relative Suspension Growth (RTG), Calculation of Mutation Frequency (MF), and total compound toxicity data (formulas and calculations are shown in Figure 1). - Evaluation criteria:
- The following criteria were used as guidelines in judging the significance of the activity of the test material in this system. In evaluating the results, it is considered that increases in mutant frequencies, which occur only at highly toxic concentrations, may be due to epigenetic events. Unfortunately, it was impossible to formulate criteria which would apply to all types of data which may be generated and therefore the conclusion of the study was based on the scientist’s evaluation.
Positive- if there was a positive dose response and one or more of the three highest doses exhibited a mutant frequency which was 2-fold greater than the background level.
Equivocal- if there was no dose response but any one or more doses exhibited a 2-fold increase in mutant frequency over background level.
Negative- if there was no dose response and none of the test cultures exhibited mutant frequency which was 2-fold greater than the background level.
Criteria for determination of a valid test
The mutation frequency of the positive controls must be at least twice that of the appropriate solvent control cultures.
The spontaneous mutation frequency of the solvent control cultures must be between 0.2 and 1.0 per 10E04 surviving cells.
The plating efficiency of the solvent controls must be greater than 50%. - Statistics:
- No data available.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- L5178Y TK+/-3.7.2c
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Remarks:
- L5178Y TK+/-3.7.2c
- Metabolic activation:
- with
- Genotoxicity:
- ambiguous
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- The test substance produced an equivocal response in the presence of exogenous metabolic activation and a negative response in the absence of metabolic activation.
- Executive summary:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK+/-, locus of the L5178Y mouse lymphoma cell line in the presence and absence of Aroclor induced rat liver S9. The S9 activated cultures were cloned over a range of test article concentrations which produced from 22% to 117% total growth.
The highest test article concentration cloned in the S-9 activated culture exhibited a mutant frequency which was more than twice the mean mutant frequency of the solvent controls. None of the nonactivated cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls.
The results indicated that, under the conditions of this test, test material produced a negative response in the absence of exogenous metabolic activation and an equivocal response in the presence of metabolic activation.
Three assays were conducted on the test material in the presence of S9 as follow-up studies to that reported above. In the first assay, the cultures that were cloned were treated with a range of test article concentrations which produced from 3% to 71% total growth. There was some contamination in this assay and complete results were obtained for 11 of the 18 cultures that were cloned. However, all the 11 cultures exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The assay was repeated due to the contamination and the erratic dose-response relationship in toxicity in the treated cultures. All the cultures that were cloned exhibited mutant frequencies which were more than twice the mean mutant frequency of the solvent controls. Previous studies with the test material had also demonstrated a precipitous toxic response. In the second experiment, the cultures that were cloned were treated with a range of concentrations which produced from 3% to 44% total growth. 7 of the 7 cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. In a third experiment, conducted concurrently with an assay on Calcium Dialkyl Dithiophosphate and with a second sample of test material, the cultures that were cloned were treated with a range of concentrations which produced from 27% to 96% total growth. None of the cultures that were cloned exhibited mutant frequencies which were significantly greater then the mean mutant frequency of the solvent controls. However, a dose-dependent response was noted.
- Endpoint:
- in vitro transformation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 24 Nov 1982 - 24 Sept 1984
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: Schechtman, L., Kouri, R., Progress in Genetic Toxicology, Elsevier/North-Holland Biomedical Press, New York, pp. 307-316 (1977).
- Deviations:
- no
- Principles of method if other than guideline:
- The BALB/3T3 transformation assay was designed to allow the expression of transformed foci of high cell density and aberrant cell morphology on a confluent monolayer of non-transformed, contact-inhibited 3T3 cells. This system has been demonstrated to be sensitive to the transforming activity of a variety of chemicals.
The non-activated assay was performed by exposing BALB/3T3 cells, in monolayer, to three concentrations of the test article as well as positive and negative controls for 24 hours in the absence of a supplemental exogenous mammalian metabolic activation system, after which the cells were cultured for estimation of the cytotoxic effects of treatment and the induction of phenotypic transformation. - GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell transformation assay
- Target gene:
- Not applicable
- Species / strain / cell type:
- mammalian cell line, other: BALB/3T3 clone A31 cell line.
- Details on mammalian cell type (if applicable):
- - Type and identity of media: Eagle’s Minimal Essential Medium supplemented with 10% FBS, 1% L-glutamine, and 2% penicillin-streptomycin.
- Properly maintained: no data available.
- Periodically checked for Mycoplasma contamination: no data available.
- Periodically checked for karyotype stability: no data available.
- Periodically "cleansed" against high spontaneous background: not applicable. - Additional strain / cell type characteristics:
- other: This cell line is characterized by post confluence inhibition of proliferation and the ability to form distinct quantifiable foci of aberrant cell morphology following exposure to chemical carcinogens.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9: 9000 x g supernatant of Aroclor 1254 induced Fischer 344 rat liver homogenate
- Test concentrations with justification for top dose:
- Without metabolic activation: 8, 15, 30 µg/mL
With metabolic activation Study A: 4, 5, 6 µg/mL
With metabolic activation Study B: 6, 7, 8 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test substance was solubilized in acetone. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Benzo(a)pyrene and N-methyl-N-nitro-N-nitrosoguanidine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: none.
- Exposure duration: 24 h
- Expression time (cells in growth medium): 4-6 weeks (transformation assay); 7-10 days (cytotocicity).
NUMBER OF REPLICATIONS: triplicates for cytotoxicity; 12-15 replicates for phenotypic transformation assay.
NUMBER OF CELLS EVALUATED: seeded at 250 cells/60 mm dish for cytotoxicity; 1.00E04 cells/60 mm dish for phenotypic transformation assay.
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- The cloning efficiency of the solvent control must be equal to or greater than 25%. The relative survival of cells exposed to the test article must fall within 30-60% for one dose level and 60-90% for another dose level. This requirement may be waived in the event of a positive result, or if solubility limits preclude testing in a toxic range. The number of Type III foci in the negative control must not exceed 1 focus per total replicate dishes. The positive control must induce a significant (p<0.05) number of Type III foci relative to the negative control.
- Statistics:
- The transforming potential of each treatment condition was compared to that of the solvent control using a special application of the Poisson distribution as follows:
The proportion (P) of all cells not subject to induced transformation is equal to the number of surviving cells in the solvent control group divided by the total of the surviving cells in the solvent control and treatment group. Significance is determined by p = [n!/n! (n-n1)!]*p^n1 *(1-p) ^(n-n1). P is the probability, n is the number of induced Type II foci, n1 is the number of spontaneous type II foci. The induced transformation frequency is considered significant if the p = 0.05. - Key result
- Species / strain:
- other: BALB/3T3 cell line
- Metabolic activation:
- without
- Genotoxicity:
- other: transforming activity was not observed
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- other: BALB/3T3 cell line
- Metabolic activation:
- with
- Genotoxicity:
- other: transforming activity was observed
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- BALB/3T3 cell transforming activity was not observed under the conditions of this study in the absence of metabolic activation. BALB/3T3 cell transforming activity was observed under the conditions of this study in the presence of metabolic activation.
- Executive summary:
RESULTS AND DISCUSSION:
The morphological transformation potential of test material was conducted using BALB/3T3 cell line. The following results were obtained.
Without S-9:
In the absence of metabolic activation (-S9), relative to solvent control, cell survival was 49%, 66% and 95% at 30, 15 and 8.0 µg/mL respectively. No Type II or Type III transformed foci were observed at any dose level tested. One spontaneous Type III focus was observed in the solvent control. The positive control induced 6 Type II and 17 Type III foci. Based on these results the negative and positive controls fulfilled the requirements for the determination of a valid test.
With S-9:
In the presence of metabolic activation (+S9), relative to solvent control, cell survival was 65%, 79% and 95% at 6, 5 and 4 µg/mL respectively (Study A) and 0%, 0% and 55% at 8, 7, and 6 µg/mL (Study B). A repeat assay was performed in an attempt to obtain higher levels of toxicity. Type II and Type III foci were observed in the treated cultures as follows:
Initial Assay (+S9)
4 µg/mL: 1 Type II; 3 Type III
5 µg/mL: 5 Type II; 4 Type III
6 µg/mL: 2 Type II; 1 Type III
Repeat Assay (+S9)
6 µg/mL: 0 Type II; 2 Type III
7 µg/mL: toxic
8 µg/mL: toxic
The transformation frequency of the 5 µg/mL dose group was statistically significantly greater than the solvent control.
No spontaneous Type III foci were observed in the solvent control in the initial or repeat assay. The positive control induced 9 Type II and 6 Type III foci in the initial assay and 6 Type II and 7 Type III foci in the repeat assay. Based on these results the negative and positive controls fulfilled the requirements for the determination of a valid test.
- Endpoint:
- genetic toxicity in vitro, other
- Remarks:
- QSAR prediction
- Type of information:
- (Q)SAR
- Adequacy of study:
- supporting study
- Study period:
- 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
- Principles of method if other than guideline:
- - Software tool(s) used including version: Vega v1.1.3 and OECD QSAR Toolbox v3.4
- Model(s) used:
OECD QSAR Toolbox v3.4: DNA binding by OASIS v.1.4, DNA binding by OECD, DNA alerts for AMES by OASIS v.1.4, DNA alerts for CA and MNT by OASIS v.1.1, in vitro mutagenicity (Ames test) alerts by ISS, in vivo mutagenicity (Micronucleus) alerts by ISS, Carcinogenicity (genotox and nongenotox) alerts by ISS and Protein binding alerts for Chromosomal aberration by OASIS
Vega v1.1.3: CAESAR (Vega), SarPy/IRFMN (Vega), ISS (Vega) and KNN/Read-Across (Vega)
- Model description:
OECD QSAR Toolbox v3.4: (Q)SAR screening was done applying all profilers present in the Toolbox. The relevant ones for mutagenicity, carcinogenicity and Cramer classification were then selected. The original data is available on request.
Vega v1.1.3: Vega is a freely available software that includes (Q)SAR models for several endpoints. Four models for mutagenicity and four models for carcinogenicity were used for this evaluation. Vega also provides information about the reliability of its predictions, through the so-called Applicability Domain Index (ADI), a numerical index that ranges from 0 (molecule out of the applicability domain) to 1 (molecule in the applicability domain).
- Justification of QSAR prediction: QSAR prediction was used as supporting information. - GLP compliance:
- no
- Key result
- Additional information on results:
- Based on the results predicted by Vega v1.1.3 software, no structural alerts related to mutagenic effect have been identified by the three models including knowledge-based rules (CAESAR and ISS) or statistically identified rules (SarPy/IRFMN). The Read-Across model based its prediction on the four most similar compounds identified within the internal dataset; all of them had a similarity degree above 0.8 with the target molecules. The three most similar compounds reported for each evaluated molecule are experimentally non-genotoxic and always correctly predicted by the utilized models. The low reliability indexes reported for all models seem to be related only to the presence of one atom centered fragment (ACF) which is not present in the models’ datasets: S and P bonded by a single bond (please refer to table 1 under "any other information on results incl. tables").
Based on the results predicted by OECD Toolbox, no alerts were found in the different OECD Toolbox profilers for the target substance. For the source substance, the Carcinogenicity (genotox and nongenotox) alerts by ISS identified a structural alert possibly related to non-genotoxic carcinogenicity: “Substituted n-alkylcarboxylic acids (Nongenotox)” (please refer to table 2 under "any other information on results incl. tables").
None of the models included in VEGA or the OECD Toolbox profilers were able to identify structural alerts related to genotoxic effect in both evaluated molecules. The results of VEGA models are always out of the applicability domain, however this seems to be mostly related to the presence of an atom centered fragment (ACF) not identified within the models’ datasets: S and P bonded by a single bond. As this fragment has not been identified by any model or profiler as possibly related to genotoxicity effect, and considering that the other parameters used by VEGA to evaluate the applicability domain are compliant (or nearly compliant), the VEGA predictions can be considered valid.
The results obtained within this evaluation support the non-genotoxicity of Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts (CAS 4563-56-8). - Remarks on result:
- no mutagenic potential (based on QSAR/QSPR prediction)
Referenceopen allclose all
(1) The initial toxicity test performed on test material in the absence of S9 indicated a threshold level of complete toxicity at 0.05 µL/mL. Based on these data, the test material was tested in a mutagenesis assay in the absence of S-9 over a range of concentrations from 0.05 µL/mL to 0.00067 µL/mL. After two day expression period, 10 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.0089, 0.0067, 0.0050, 0.0038, 0.0028, 0.0021, 0.0016, 0.0012, 0.00089 or 0.00067 µL/mL test material. These concentrations produce a range in suspension growth of 37% to 91%. None of the cultures that were cloned exhibited mutant frequency which were significantly greater than the mean mutant frequency of the solvent controls. The total growth of the cultures ranged from 35% to 98%.
(2) An initial toxicity test was conducted in the presence of S9 on test material. The results indicated a threshold level of complete toxicity at 0.05 µL/mL. Based on these data, the test material was tested in a mutagenesis assay in the presence of S9 over a range of concentrations from 0.05 µL/mL to 0.00067 µL/mL. After two day expression period, 10 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.021, 0.016, 0.012, 0.0089, 0.0067, 0.0050, 0.0038, 0.0028, 0.0021 or 0.0016 µL/mL test material. These concentrations produce a range in suspension growth of 25% to 97%. One culture (0.021 µL/mL) that was cloned exhibited a mutant frequency which was 2.3 times the mean mutant frequency of the solvent controls. The total growth of this culture was 22%. None of the remaining cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The total growth of these cultures ranged from 91% to 117%.
(3) Two mutagenesis assays in the presence of S9 were conducted as follow-up studies to the original assays. In the first assay the cultures were treated in triplicate with a range of test article concentrations from 0.030 to 0.017 µL/mL. After a two day expression period, 18 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.022, 0.021, 0.020, 0.019, 0.018 or 0.017 µL/mL. These concentrations produced a range in suspension growth of 7% to 76%. All the cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The increases in mutant frequency ranged from 9.4 to 2.2 times the mean mutant frequency of the solvent controls. The total growth of the cultures ranged from 3-71%.
(4) The assay (described under (3)) was repeated due to some contamination and an erratic toxic response. The repeat assay was conducted over the same dose range. After a two day expression period, 7 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.021, 0.018 or 0.017 µL/mL. These concentrations produced a range in suspension growth of 7% to 47%. All the cultures that were cloned for which complete results were obtained exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The increases in mutant frequency ranged from 11.3 to 3.1 times the mean mutant frequency of the solvent controls. The total growth of the cultures ranged from 3-44%.
(5) A second sample of the test material was tested. The initial toxicity test indicated a complete toxicity in the presence of S9 at 0.05 µL/mL. Based on the results, the test material was tested in a mutagenesis assay in the presence of S9 over a range of concentrations from 0.05 µL/mL to 0.00067 µL/mL. After a two day expression period, 10 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.021, 0.016, 0.012, 0.0089, 0.0067, 0.0050, 0.0038, 0.0028, 0.0021 or 0.0016 µL/mL. These concentrations produced a range in suspension growth of 31% to 100%. None of the cultures that were cloned exhibited mutant frequency which were significantly greater then the mean mutant frequency of the solvent controls. The total growth of the cultures ranged from 27% to 96%. However, a dose-dependent increase in mutant frequency was noted. The increase ranged from 1.0 to 1.9 times the mean mutant frequency of the solvent controls.
Results
Without S-9:
In the absence of an exogenous metabolic activation system at doses of 30, 15 and 8 µg/mL which were selected following a preliminary dose range-finding clonal toxicity assay. Dosing solutions were prepared in acetone and then diluted to the appropriate 2x concentration with complete EMEM. The solvent control dishes were treated with acetone at a final concentration of 2 µL/mL.
Relative to the acetone control, the cell survival was approximately 49%, 66% and 95% at 30, 15 and 8 µg/mL, respectively (Table 1).
One spontaneous type III focus was observed in the solvent control for a background transformation frequency of 0.12 x 10E-04. No foci were observed in the test article-treated groups. MNNG induced 6 type II and 17 type III foci for a transformation frequency of 28.33 x 10E-04 (p < 0.01, Modified Poisson Distribution) (Table 2).
With S-9:
The test material was tested in the presence of a S-9 activation system at doses of 6, 5 and 4 µg/mL which were selected following a preliminary dose range-finding clonal toxicity assay. A repeat assay was performed at 8, 7 and 6 µg/mL in an effort to obtain higher levels of toxicity. Dosing solutions were prepared in acetone and then diluted to the appropriate 2x1 concentration with PBS. The solvent control dishes were treated with acetone at a final concentration of 2 µL/mL. The cytotoxic effects of a 2-hour treatment relative to the acetone control, the cell survival was approximately 65%, 79% and 95% at 6, 5 and 4 µg/mL, respectively (Experiment 1) and 0%, 0% and 55% at 8, 7 and 6 µg/mL, respectively (Experiment 2) (Table 3).
No spontaneous type III foci were observed in the solvent control for background transfomative frequencies of < 0.17 x 10E-04 in Experiment 1 and < 0.15 x 10E-04 in Experiment 2. In Experiment 1, two type II and one type III foci were observed at 6 µg/mL, five type II and four type III were observed at 5 µg/mL, and one type II and three type III foci were observed at 4 µg/mL. The induced transformation frequency of 0.78 x 10E-04 was statistically increased (p < 0.05, modified Poisson Distribution) relative to the acetone control. In Experiment 2, 8 and 7 µg/mL were toxic. Two type III foci were observed at 6 µg/mL. BaP induced 9 type II and 6 Type III foci (Experiment I), and 6 type II and 7 type III foci (Experiment 2) for transformation frequencies of 2.4 x 10E-04 and 3.3 x 10E-04, respectively (p < 0.01, modified Poisson Distribution) (Table 4).
Table 1: VEGA mutagenicity predictions for the target substance Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts (CAS 4563-56-8) and for and for the source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8)
Model |
Model type |
CAS |
Predicted Mutagen activity |
Reliability (ADI) |
Similarity index |
Accuracy index |
Concordance index |
Descriptors range check |
ACF index |
CAESAR (Vega) |
Hybrid (a) |
4563-56-8 |
NON-Mutagenic |
Low (0.562) |
0.877 |
1 |
1 |
TRUE |
0.6 |
4259-15-8 |
NON-Mutagenic |
Low (0.551) |
0.844 |
1 |
1 |
True |
0.6 |
||
SarPy/IRFMN (Vega) |
Statistical (b) |
4563-56-8 |
NON-Mutagenic |
Low (0.562) |
0.877 |
1 |
1 |
n.c. |
0.6 |
4259-15-8 |
NON-Mutagenic |
Low (0.551) |
0.844 |
1 |
1 |
n.c. |
0.6 |
||
ISS (Vega) |
Knowledge-based (c) |
4563-56-8 |
NON-Mutagenic |
Low (0.531) |
0.782 |
1 |
1 |
n.c. |
0.6 |
4259-15-8 |
NON-Mutagenic |
Low (0.533) |
0.79 |
1 |
1 |
n.c. |
0.6 |
||
KNN/Read-Across (Vega) |
Read-Across |
4563-56-8 |
NON-Mutagen |
Low (0.538) |
0.805 |
1 |
1 |
n.c. |
0.6 |
4259-15-8 |
NON-Mutagenic |
Low (0.541) |
0.813 |
1 |
1 |
n.c. |
0.6 |
(a) The CAESAR model is a “hybrid” model, based on statistically determined molecular descriptors as well as on knowledge-based structural alerts.
(b) The SarPy/IRFMN model includes structural alerts statistically identified using the SarPy software, as possibly related to mutagenicity, from a dataset of molecules and experimental Ames test information.
(c) The ISS model includes the knowledge-based structural alerts described by Benigni et al. (2008) and included in the ToxTree software.
(d) The KNN/Read-Across identifies the four most similar substances present within the Vega dataset and uses them to evaluate the mutagenicity of the target molecule.
Table 2: OECD Toolbox mutagenicity-related profiling of Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts (CAS 4563-56-8) and for Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8)
OECD Toolbox profiler |
CAS 4563-56-8 |
CAS 4259-15-8 |
DNA binding by OASIS v.1.4 |
No alert found |
No alert found |
DNA binding by OECD |
No alert found |
No alert found |
DNA alerts for AMES by OASIS v.1.4 |
No alert found |
No alert found |
DNA alerts for CA and MNT by OASIS v.1.1 |
No alert found |
No alert found |
in vitro mutagenicity (Ames test) alerts by ISS |
No alert found |
No alert found |
in vivo mutagenicity (Micronucleus) alerts by ISS |
No alert found |
No alert found |
Carcinogenicity (genotox and nongenotox) alerts by ISS |
No alert found |
Structural alert for nongenotoxic carcinogenicity|Substituted n-alkylcarboxylic acids (Nongenotox) |
Protein binding alerts for Chromosomal aberration by OASIS |
No alert found |
No alert found |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Micronucleus test: not clastogenic in CD-1 mice
RA from CAS 4259-15-8
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- GLP compliance:
- yes
- Type of assay:
- other: cytogenicity / erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- CD-1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 8 weeks and 2 days
- Weight at study initiation: 29.8-37.8 g (male) and 23.3-28.8 g (female)
- Assigned to test groups randomly: yes
- Housing: five per cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12 hours - Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: Peanut oil
- Amount of vehicle (if gavage or dermal): 10 mL - Duration of treatment / exposure:
- 24, 48, and 72 h
- Frequency of treatment:
- Once
- Dose / conc.:
- 6 mg/kg bw/day (nominal)
- Dose / conc.:
- 12 mg/kg bw/day (nominal)
- Dose / conc.:
- 24 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide
- Vehicle: sterile water
- Doses / concentrations: 60 mg/kg bw - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
Range finding study performed to find the maximum tolerated dose
DETAILS OF SLIDE PREPARATION:
Slides fixed with methanol and stained in May-Grunwald solution followed by Giemsa.
METHOD OF ANALYSIS:
Scored for micronuclei and the polychromatic erythrocyte (PCE) to normochromatic erythrocyte (NCE) cell ration. - Evaluation criteria:
- Statistically sifnificant dose-related increase in micronucleated PCE's and the detection of a statistically sifnificant positive response for at least one dose level.
- Statistics:
- The frequency of micronucleated polychromatic erythrocytes between treated groups and vehicle controls were compared. Tests included Cochran-Armitage test for trend, a one-way analysis of variance and Dunnett’s procedure.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Under the conditions of this study, the frequency of micronucleated polychromatic erythrocytes was not increased in treated mice when compared with the control group.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Justification for read-across
There are no reliable experimental data available regarding genetic toxicity for Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts, neutral and basic (CAS 4563-56-8). Read-across from an appropriate substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VII-VIII, 8.4. Common functional groups, structural similarities and comparable toxicological properties of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).In addition,QSAR evaluation for genotoxicity using OECD QSAR Toolbox v3.4 and Vega v1.1.3 mutagenicity models with Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts and Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) was performed.
In vitro gene mutation study in bacteria: CAS 4259-15-8
Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8)was investigated for mutagenicity in bacteria (Ames test) in a study similar to OECD guideline 471 under GLP conditions (Key, 1997). The Salmonella typhimurium strains TA 1535, TA 1537, TA 100 and TA 98 were exposed to concentrations ranging from 25 - 5000 µg/plate and from 1 – 500 µg/plate in ethanol with and without the addition of a metabolic activation system, respectively, in two independent assays (plate incorporation). Cytotoxicity was observed in all tester strains with and without metabolic activation (not further specified).Based on the results of the conducted study, the test substance did not show mutagenic properties in S. typhimurium TA98, TA100, TA1535 and TA 1537 with and without metabolic activation.
In vitro gene mutation study in mammalian cells: CAS 4259-15-8
In vitro mammalian gene mutation potential at thymidine kinase (TK) locus was measured using L5178Y mouse lymphoma cell line after treatment with various concentrations of Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) (Key, 1983). 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. In the absence of metabolic activation, the test material did not cause mutagenic activity. In the presence of S9 microsomal enzyme, 4 independent tests were conducted on two samples of the test material: in the first study (0.0016 – 0.021 µL/mL), the highest concentration (0.021 µL/mL) exhibited an increase of the mutant frequency. The second study was disqualified due to contamination. The third experimental cultures were treated with concentrations (0.017 - 0.021 µL/mL) which produced total growth range from 44 to 3%. The increases in mutant frequency ranged from 3.1 to 11.3 times the mean mutant frequency of the solvent controls. In the fourth experiment, a second sample of the test substance was used. The cultures were treated with concentrations ranging from 0.0016 to 0.021 µL/mL which resulted in a total growth from 96 to 27%. None of the cultures that were cloned exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. However, a dose-dependent increase in mutant frequency was noted. The increase ranged from 1.9 to 1.0 times the mean mutant frequency of the solvent controls.
Based on the results of this study, the test substance was considered to be non-mutagenic in the absence of a metabolic activation system and considered to be ambiguous in the presence of a metabolic activation system due to a lack of reproducibility between repeated experiments, and due to the observed mutagenic activity which was concurrent with the presence of extensive cytotoxic damage at high doses.
BALB/3T3 Transformation Test: CAS 4259-15-8
A supporting study on the BALB/3T3 transformation with Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) was included (Key, 1984). Based on the results of the conducted study, the test substance demonstrated transformation activity with S-9 activation. However, statistically significant increases in transformation frequencies occurred only at the highest tested dose which was associated with noticeable cytotoxicity. Without S-9 activation, an increase in transformation frequencies was not observed.
Mouse Micronucleus Test (in vivo): CAS 4259-15-8
An in vivo mammalian cell gene cytogenicity assay according to OECD guideline 474 and under GLP was performed withZinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8)in male and female CD-1 mice (Key, 1997). The maximum tolerated dose was selected based on the results of a range finding study, thus the animals were exposed to doses of 6, 12 and 24 mg/kg bw/day. Control animals treated with the solvent (peanut oil) and the positive control cyclophosphamide were included in the study. Toxicity was observed (not further specified) and the positive and vehicle control were considered to be valid. An increase of micronucleated polychromatic erythrocytes was not observed in any of the dose groups, thus, the test substance was not clastogenic in CD-1 mice.
(Q)SAR using Vega v1.1.3 and OECD QSAR Toolbox v3.4
None of the models included in VEGA or the OECD Toolbox profilers were able to identify structural alerts related to genotoxic effect in the evaluated target and source substance. The results of VEGA models are always out of the applicability domain, however this seems to be mostly related to the presence of an atom centered fragment (ACF) not identified within the models’ datasets: S and P bonded by a single bond. As this fragment has not been identified by any model or profiler as possibly related to genotoxicity effects, and considering that the other parameters used by VEGA to evaluated the applicability domain are compliant (or nearly compliant), the VEGA predictions can be considered valid.
The results obtained within this evaluation support the non-genotoxicity of Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts, neutral and basic (CAS 4563-56-8).
Conclusion
Overall, the source substance Zinc bis[O,O-bis(2-ethylhexyl)] bis(dithiophosphate) (CAS 4259-15-8) was considered to be not mutagenic in bacterial cells with and without metabolic activation, to benon-mutagenic in the absence of a metabolic activation system and to be ambiguous in the presence of a metabolic activation system in the mouse lymphoma assay. Moreover, the source substance showed a transforming activity in the presence of S9 mix in the BALB/3T3 transformation assayat the highest tested dose which was associated with noticeable cytotoxicity. In an in vivo mouse micronucleus test no clastogenic properties were determined after treatment of male and female CD-1 mice with the source substance. QSAR analysis performed with the target and the source substance revealed non-mutagenic and non-genotoxic properties in all models used. Therefore, taken into account all available data and based on the analogue approach, the source and the target substance can be considered as non-mutagenic in bacterial and mammalian cells and as non-clastogenic in vivo.
Justification for classification or non-classification
Applying the RA-approach, the available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.
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.