Zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study with the source substance CAS 84605-29-8. Comparable with standardised tests, scientifically accepted methods, and is sufficiently detailed. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1983

Report date:

1984

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

270-608-0

EC Name:

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

Cas Number:

68457-79-4

Molecular formula:

Too complex

IUPAC Name:

zinc bis(O,O-diisobutyl dithiophosphate)

Details on test material:

- Name of test material (as cited in study report): T-1855
- Substance type: organic
- Physical state: clear amber, slightly viscous liquid

Method

Target gene:

The thymidine kinase, TK +1-, locus of the L5178Y mouse lymphoma cell line.

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: no data available.
- 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 (in-house male Sprague-Dawley rates). Ip injections with 2:1 Aroclor 1242 : Aroclor 1254 (in corn aoil at 200 mg/mL). 5 days post injection, livers were excised.

Test concentrations with justification for top dose:

(without S-9): 0.013, 0.010, 0.0075, 0.0056, 0.0042, 0.0032, 0.0024, 0.0018, or 0.0013 µl/ml
(with S-9): 0.024, 0.018, 0.013, 0.010, 0.0075, 0.0056, 0.0042, 0.0032, 0.0024, or 0.0018 µl/ml.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: No data available.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulphonate AND 7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

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 concentration, 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 fog 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 104 surviving cells.
The plating efficiency of the solvent controls must be greater than 50%.

Statistics:

No data available

Results and discussion

Test results

Test results 1

Species / strain:

other: L5178Y TK+/-3.7.2c mouse lymphoma cell line

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

Test results 2

Species / strain:

other: L5178Y TK+/-3.7.2c mouse lymphoma cell line

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: L5178Y TK+/-3.7.2c mouse lymphoma cell line

Any other information on results incl. tables

1. The initial toxicity test performed on test material in the absence of S-9 indicated a threshold level of complete toxicity at 0.1 ul/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 0.01 ul/ml to 0.0013 ul/ml. After two day expression period, 10 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with 0.013, 0.010, 0.0075, 0.0056, 0.0042, 0.0032, 0.0024, 0.0018, or 0.0013 ul/mltest material. These concentrations produced a range in suspension growth of 11% to 96%. Two of the cultures (0.013 and 0.010 ul/ml) that were cloned exhibited mutant frequencies which were 10.8 and 2.5 time respectively, the mean mutant frequency of the solvent controls. The total growth of the cultures was 2% and 4%. None of the remaining 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 20% to 92%.

 

2. An initial toxicity test was conducted in the presence of S-9 on test material the results indicated a threshold level of complete toxicity at 0.05 ul/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 0.1 ul/ml to 0.0013 ul/ml. After two day expression period, 10 cultures were cloned based on their degree of toxicity. The cultures that were cloned were treated with0.024, 0.018, 0.013, 0.010, 0.0075, 0.0056, 0.0042, 0.0032, 0.0024, or 0.0018 ul/mltest material. These concentrations produce a range in suspension growth of 11% to 82%. One culture that was cloned (0.024ul/ml) exhibited mutant frequency which was 7.2  times the mean mutant frequency of the solvent controls. The total growth of the cultures was 6%. None of the remaining 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 77% to 125%.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material produced a negative response in the presence and absence of exogenous metabolic activation.

Executive summary:

The study was conducted according to a method that was designed to assess the potential mutagenecity 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 S-9.

The nonactivated cultures were cloned over a range of test article concentrations which produced from 47% to 123% total growth in one assay and from 2% to 92% total growth in a second assay. The S-9 activated cultures were cloned over a range of test article concentrations which produced from 6% to 125% total growth.

The highest test article concentrations cloned in the S-p activated cultures exhibited a mutant frequency which was more than twice the mean mutant frequency of the solvent controls. Two of the nonactivated culture were significantly greater than the mean mutant frequency of the solvent controls. These results are not considered significant as the total growth of these cultures was less than 10%. TFR resistance observed at these highly toxic levels may be due to epigenetic events.

Conclusion

The results indicated that, under the conditions of this test, test material produced a negative response in the presence and absence of exogenous metabolic activation. In the presence of metabolic activation, the total growth of the treated cultures that were cloned did not cover the critical range of survival (10-40%). A precipitous toxic response was induced by the test article. The cultures treated with the two highest concentrations of test article had 6& and 77% total growth. It was felt that a repeated assay would not provide any additional information since the difference in concentration between the cultures having 6% and 77% total growth was only 0.006 ul/ml.