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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted on structural analogue and suitable for read across; Comparable with standardised tests, scientifically accepted methods, and is sufficiently detailed

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

IUPAC Name:

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

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 ul/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 ul/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

EC 270-608-0:

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%.

 

 

JUSTIFICATIONS FOR READ-ACROSS

Mutagenicity potential in mammalian cells was not evaluated for EC 272-723-1, however experimental data on structurally related substance EC 270-608-0 is available and suitable for read-across. Justifications for read-across:

Manufacture/Usage:

EC 272-723-1 and EC 270-608-0 are generically referred to as zinc dialkylthiophosphate (ZDDP) which are produced under similar manufacturing procedures and used in commerce as multi-functional anti-wear and anti-oxidation inhibitor performance components in passenger motor oils, diesel engine oils and industrial oils such as hydraulic lubricants.

Chemical Similarity:

EC 272-723-1 and EC 270-608-0 consist of alkyl substituted phosphorodithioic acid structures complexed with zinc.

EC 272-723-1:Phosphorodithioic acid, mixed O,O-bis(2-ethylhexyl and iso-propyl) esters, zinc salts, referred to as “mixed 2-ethylhexyl and isopropyl derivative”.

EC 270-608-0:Phosphorodithioic acid, mixed O,O-bis(iso-butyl and pentyl) esters, zinc salts, referred to as “mixed isobutyl and pentyl derivative”.

These ZDDPs share similar core structures - alcohol ester of dithiophosphate, specific structural variations that relate to their alcohol group substituent are the alkyl chain length and the degree of branching of the alcohol charge. Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model chemical structures of the analogues showed comparable values for relevant molecular descriptors (e.g., number of H bond acceptor atoms), and gave a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical; peer reviewed literature indicates that values greater than 0.6 are significantly similar); therefore chemical structures of the analogues have determined to be sufficiently close for there to be a reasonable expectation of similar toxicological effects.

Physicochemical Properties:

Standard physicochemical properties for each substance were listed in Table 1.

Table 1. Establishment of similarity between the data donating substance and the data accepting substance

EC	Alkyl group	Average MW	log Kow	Water Sol (ppm)
270-608-0 (donate data)	C4, branched C5, mixture of linear and branched	576	0.69	1658
272-723-1  (accept data)	C3/C8 branched	632	0.84	2111

 

As shown in Table 1, EC 270-608-0 and EC 272-723-1 showed small differences in molecular weight, Log Kow and water solubility, therefore, read across between these two substances are feasible.

Biologically Active Functional Groups:

The ester group presents in each of the analogue members, and is expected to exhibit similar biological activities. Non-random patterns were observed for the toxicological effects(e.g. available data showed low levels of acute toxicity effect, lacking of mutagenic effect in bacteria, lacking of cryptogenic effects in rats, consistent trend of change in ecotoxicity effect, etc.), these common behaviors and consistent trends suggest a common mechanism/mode of action, with little influence from the length of carbon chain. These facts further supported read-across between the analogue members.

Available Data and Adequacy for Read-across:

EC270-608-0: negative in L5178Y tk^+/-mouse lymphoma mutagenesis assay (with and without metabolic activation).

By read across to this study, EC 272-723-1 was predicted to be not mutagenic in mammalian cells.

Conclusion:

In vitromammalian cell gene mutation assays have been conducted for EC 270-608-0. Based on the abovementioned justifications, results from EC 270-608-0 was used to fill this data gap for EC272-723-1, and EC 272-723-1 was predicted to be non-mutagenic in mammalian cells.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

EC 270-608-0:
The test material produced a negative response in the presence and absence of exogenous metabolic activation.
EC 270-723-1:
Results from EC 270-608-0 was used to fill this data gap for EC272-723-1, and EC 272-723-1 was predicted to be non-mutagenic in mammalian cells.

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.