Phosphorodithioic acid, O,O-di-dodecyl-esters, zinc salts, neutral and basic

Endpoint summary

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

Reference

Reference 1

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.

Reference 2

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

(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.

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.

 

Reference 3

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

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).

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.

 

Reference 4

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)

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

Reference

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.

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.