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

Endpoint summary

Additional information

Justification for grouping of substances and read-across

There are no data available on the potential for induction of gene mutations in mammalian cells of zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate). In order to fulfil the standard information requirements set out in Annex VIII, Section 8.4.3, in accordance with Annex XI, Section 1.5 of Regulation (EC) No 1907/2006, read-across from structurally similar substances is conducted.

In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across).

Having considered the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006, whereby physicochemical, toxicological and ecotoxicological properties may be predicted from data for reference substance(s) by interpolation to other substances on the basis of structural similarity, Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts (CAS 68457-79-4) and Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts (CAS 84605-29-8) are selected as reference substances for assessment of gene mutations in mammalian cells.

The read-across is based on structural similarity as all read-across chemicals belong to the substance class of Zinc alkyldithiophosphates (ZDDP) which are produced by a chemical reaction of Phosphorous pentasulfide with low molecular weight alcohols followed by a subseuqent reaction with zinc oxide. A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

Overview of genetic toxicity

	Target substance (a)	Source substance 1 (b)	Source substance 2
CAS No.	6990-43-8	68457-79-4	84605-29-8
Chemical name	zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate)	Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts	Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts
In vitro gene mutation in bacteria	Experimental result: not mutagenic	--	--
In vitro cytogenicity in mammalian cells	--	--	--
In vitro gene mutation in mammalian cells	RA: CAS 68457-79-4 RA: CAS 84605-29-8	Experimental result: not mutagenic	Experimental result: not mutagenic
In vivo cytogenicity	Experimental result: not clastogenic	--	--

(a) The substance subject to the REACh Phase-in registration deadline of 31 May 2013 is indicated in bold font. Only for this substance a full set of experimental results and/or read-across is given.

(b) Reference (read-across) substance(s) are indicated in normal font. Lack of data for a given endpoint is indicated by “--“.

 

In vitro gene mutation in bacteria

CAS 6990-43-8

A reverse mutation assay (Ames test) was conducted with zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) according to a test method similar to OECDC guideline 471 (Brusik, 1976). The tester strains Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and Saccharomyces cerevisiae D4 were treated with the undiluted test item using the plate incorporation method at 0.001, 0.01, 0.1, 1.0 and 5.0 µL/plate, without replications, both with and without metabolic activation (S9 mix). Positive and DMSO (vehicle for positive control substances) controls were included. A repeat experiment was carried out with the tester strain TA 100 with metabolic activation.

Except for the tester strain TA 1538, cytotoxic effects (decrease in the number of revertant colonies) were observed in the S. typhimurium and S. cerevisiae strains. No increases in the frequency of revertant colonies were recorded for any of the bacterial and yeast strains at any concentration either with or without metabolic activation. The DMSO and positive control treated plates yielded the expected results.

Under the conditions of this test, the test item was considered to be non-mutagenic in bacteria and yeast.

In vitro gene mutation in mammalian cells

CAS 68457-79-4

The substance Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts was tested in an in vitro mammalian cell mutation assay according to a test method similar to OECD guideline 476 (Rogers-Back, 1983). Mouse lymphoma L5178Y cells were treated with acetone (vehicle control) or the test substance at 0.0013-0.013 µL/mL without metabolic activation and at 0.0018-0.024 µL/mL with metabolic activation (S9-mix). Cells were treated in triplicates for 4 h. Ethylmethanesulphonate (-S9 mix) and 7,12-dimethylbenzanthracene (+S9 mix) were included as positive controls. The highest concentrations with and without metabolic activation were selected based on a preliminary toxicity test, in which the test substance showed a threshold level of complete toxicity at 0.1 (-S9-mix) and 0.05 (+S9-mix) µL/mL.

Increasing cytotoxicity was observed both in the absence and presence of S9 mix. Without metabolic activation, cultures were cloned over a range of test item concentrations which produced from 123 to 47% total growth in one assay and from 92 to 2% total growth in a second assay. In the presence of metabolic activation, cultures were cloned over a range of test item concentrations which produced from 125 to 6% total growth.

The highest concentrations in the presence of S9-mix resulted in mutant frequencies which were more than twice the mean mutant frequency of the solvent controls. Two of the cultures without S9-mix showed a significantly greater mutant frequency 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%. TFT resistance observed at these highly toxic levels may be due to epigenetic events.

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 77 and 6% total growth, respectively. It was considered that a repeat assay would not provide any additional information since the difference in concentration between the cultures having 77 and 6% total growth was only 0.006 µL/mL.

It was thus concluded that the test material produced a negative response in the presence and absence of exogenous metabolic activation.

CAS 84605-29-8

An in vitro mammalian cell gene mutation assay was carried out with the substance Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts similarly to OECD guideline 476 (Rogers-Back, 1983). Mouse lymphoma L5178Y cells were treated for 4 h with the solvent (acetone) and the test item at 0.00056-0.01 µL/mL and 0.00067-0.0067 in the absence and 0.005-0.031 µL/mL in the presence of metabolic activation (S9-mix). Ethylmethanesulphonate (-S9 mix) and 7,12-dimethylbenzanthracene (+S9 mix) were included as positive controls. The highest concentrations with and without metabolic activation were selected based on a preliminary toxicity test, in which the test substance showed a threshold level of complete toxicity at 0.05 µL/mL (with and without S9-mix).

An increase in cytotoxicity was observed both in the absence and presence of metabolic activation. In the absence of metabolic activation, cultures were cloned over a range of test item concentrations which produced from 101 to 6% total growth. In the presence of S9-mix, cultures were cloned over a range of test article concentrations which produced from 88 to 1% total growth. The highest test article concentration without metabolic activation induced a mutant frequency which was more than twice the mean mutant frequency of the solvent controls. The result was not considered significant since mutant frequencies observed at such highly toxic levels may be due to epigenetic events. With metabolic activation, four cultures 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 a positive response in the presence of metabolic activation.

In vivo micronucleus test

CAS 6990-43-8

An in vivo micronucleus test was conducted with zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) in a GLP-compliant study similar to OECD guideline 474 (Sansebastian, 1988). In a preliminary dose range finding study, the test item was administered orally to 5 groups of CD-1 mice at dose levels of 250, 500, 1000, 2000 and 5000 mg/kg bw. Pharmacotoxic effects were observed at all levels and mortality was observed at all doses except 500 mg/kg bw. Due to the observed effects, the highest dose level selected for the micronucleus test (MNT) was 500 mg/kg bw as an estimate of the maximum tolerated dose with two additional lower dose levels of 100 and 250 mg/kg bw.

In the MNT, nine groups of mice were given single doses of the test material by oral gavage at 100, 250 and 500 mg/kg bw and sacrificed at 30, 48 or 72 h post-dose. Concurrent vehicle (corn oil) control groups were included for each sacrifice time point. An additional positive control group received cyclophosphamide (CP) at 60 mg/kg and was sacrificed at 30 h post-dose. Slides were prepared from the femoral bone marrow and stained. Coded slides were scored for the number of polychromatic erythrocytes (PCE) with micronuclei in 1000 PCE/animal. The ratio of polychromatic to normochromatic erythrocytes (NCE) per 1000 erythrocytes was determined for each animal.

Statistical analyses of data did not indicate a significant increase in the number of micronucleated PCEs in the treated groups compared to the corresponding vehicle control groups.

In conclusion, the test material was deemed negative for clastogenic effects at all dose levels and time intervals evaluated under the experimental conditions of the study.

Dominant Lethal Assay

A Dominant Lethal Assay was performed with zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) in rats following OECD guideline 478 and under GLP conditions (Sorg, 1988). The purpose of the study was to determine the potential of the test item to produce dominant lethal mutations in germ cells of male rats pre-treated with the test item. Based on the results of a previous range finding study, the test item was evaluated at 10, 33 and 100 mg/kg bw.

Groups of 15 (20 for high dose) young adult male Sprague-Dawley rats were administered the test item at 10, 33 and 100 mg/kg bw/day by oral gavage. Two groups of 15 animals each receiving corn oil (5 mL/kg bw/day) or triethylenemelamine (TEM, 0.05 mg/kg bw/day) were included as vehicle and positive control, respectively. Animals were treated once daily, 5 days/week for 10 weeks to cover the duration of the entire spermatogenic cycle. Animals were observed for pharmacotoxic signs before and immediately after dosing, on non-dosing days and during the mating period. Signs were observed at all dose levels primarily including post-dose salivation and decreased activity. Four males died during the dosing period. One male in the 33 mg/kg bw/day group died on Day 42. The other three males were in the 100 mg/kg bw/day group and died on Days 2, 43 and 57, respectively.

Following dose administration on the final day of dosing, males were cohoused with 2 untreated virgin young adult Sprague-Dawley female rats. After one week of mating, these females were removed and a second week of mating was initiated with two additional females. Mid-week of each cohousing was considered the time of conception and 14 days later the females were sacrificed and the contents of the uteri examined.

No statistically significant reductions in fertility or increases in pre-implantation loss or post-implantation loss were detected in females mated with treated males at any dose level. Positive control males gave a significant increase in the proportion of dead implants in their resulting pregnancies, confirming the dominant lethal effect of TEM.

Based on these results, the test item was determined to be negative in its ability to induce dominant lethal mutations at the dose levels evaluated under the conditions of the study.

Conclusions for genetic toxicity

The substance zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) has been tested in a reverse mutation assay (Ames test) in bacteria and yeast, an in vivo micronucleus test in mice and a dominant lethal assay in rats. Under the conditions of the corresponding studies, the substance was considered to be not mutagenic in bacteria and yeast, not clastogenic in mice and not able to induce dominant lethal mutations in rats.

Therefore, the available data on the structurally similar substances Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts and Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts were used for hazard assessment by mean of read-across.

Both substances have been tested in Mouse Lymphoma Assays. Phosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc salts was tested negative for the induction of gene mutations both in the presence and absence of metabolic activation, while Phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-Pr) esters, zinc salts was tested negative in the absence and positive in the presence of metabolic activation. In both studies, the test substances were highly cytotoxic.

Taken together, based on the available in vitro and in vivo data the substance zinc O,O,O',O'-tetrabutyl bis(phosphorodithioate) is considered to be not mutagenic in vitro and not clastogenic in vivo.

Justification for selection of genetic toxicity endpoint
Hazard assessment is conducted both on the basis of substance specific data and by means of read-across from a structural analogue. No study was selected, since all available in vitro and in vivo genetic toxicity studies were negative. All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties between source and target substance and overall quality assessment (refer to the endpoint discussion for further details).

Short description of key information:
Based on substance specific data and/or read-across from a structurally similar substance:
In vitro gene mutation in bacteria: negative with and without metabolic activation
In vitro gene mutation in mammalian cells: negative with and without metabolic activation (based on read-across)
In vivo cytogenicity: negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on substance specific data and read-across from a structurally similar substance following an analogue approach, the available data on the genetic toxicity of the substance do not meet the classification criteria according to Regulation (EC) No 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.