Dibutyl [(dipropoxyphosphinothioyl)thio]succinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester was considered to be non-mutagenic under the conditions of this test (SafePharm Laboratories, 1997).

These results indicate the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system (BioReliance, ****).

Under the conditions of the assay the test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay (BioReliance, ****).

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

Study period:

09 December 1996 to 20 February 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Description: Colourless liquid
- Storage: Ambient <25°C under artificial light

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 Rat Liver, induced with S9-mix

Test concentrations with justification for top dose:

Mutagenicity Assay: 0, 50, 150, 500, 1500 and 5000 μg.
5000 μg is the standard top dose recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration.

Preliminary Toxicity Assay: Conducted at dose levels of : 0, 50, 150, 500, 1500 and 5000 μg per plate.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2 aminoanthracene (in the presence of S9 Mix)

Details on test system and experimental conditions:

Preparation of Tester Strain
Overnight cultures were prepared from the appropriate frozen permanent stock. Following inoculation, each flask was placed in a shaker/incubator and incubated at 37±2°C for approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Exogenous Metabolic Activation
The S9 metabolic activation system was purchased commercially from MolTox (Boone, NC) and stored at 60°C or colder until use. It was prepared from male Sprague-Dawley rats that were injected intraperitoneally with Aroclor™ 1254. Each bulk preparation was assayed for its ability to metabolize benzo(a)pyrene and 2 aminoanthracene to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared on the day of use and contained: S9 (10%), sodium phosphate buffer (pH 7.4; 100 mM), MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM), and β-nicotinamide-adenine dinucleotide phosphate (4 mM). The Sham mix, containing 100 mM phosphate buffer at pH 7.4, was also prepared on the day of use.

Frequency and Route of Administration
The test system was exposed to the test substance via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983).

Preliminary Toxicity Assay to Select Dose Levels
The preliminary toxicity assay was used to establish the dose range over which the test substance would be assayed. TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone and ten dose levels of the test substance, with a single plate/condition, on selective minimal agar in the presence and absence of S9 mix. Dose levels for the mutagenicity assay were based upon post-treatment toxicity.

Mutagenicity Assay
TA98, TA100, TA1535, TA1537 and WP2 uvrA were exposed to the vehicle alone, positive controls and at least five dose levels of test substance, in triplicate, in the presence and absence of S9 mix.
To confirm the sterility of the S9, Sham mixes, test substance and the vehicle, each was plated on selective agar with an aliquot volume equal to that used in the assay and incubated under the same conditions as the assay.

One half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain (cells seeded) and 50.0 µL of vehicle, positive control, or test substance dilution were added to 2.0 mL of molten selective top agar at 45±2°C, vortexed, and overlaid onto minimal bottom agar. After the overlay solidified, the plates were inverted and incubated for 48 to 72 hours at 37±2C. Plates that were not counted immediately following the incubation period were stored at 2 8C until colony counting could be conducted.

Scoring
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate. Colonies were enumerated either by hand or by machine.

Tester Strain Verification
On the day of use in each assay, all tester strain cultures were checked for the appropriate genetic markers.

Key result

Species / strain:

other: other: TA1537, TA98, TA1535, TA100, and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Assay
The dose range used in the preliminary toxicity study was 0, 50, 150, 500, 1500 and 5000 μg per plate. The test material was non-toxic to the strains of bacteria used (Ta100 and WP2uvrA).

Mutagenicity Assay
Precipitate was observed at and above 1500 μg per plate; this did not interfere with the scoring of revertant colonies.

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction were found to be satisfactory.

Conclusions:

The test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Executive summary:

The test item was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. DMSO    was used as the vehicle.

 

In the preliminary toxicity assay, the dose levels tested were 0, 50, 150, 500, 1500 and 5000 μg per plate. Based upon these results, the maximum dose tested in the mutagenicity assay was 5000 μg per plate.

 

In the mutagenicity assay, the dose levels tested were 50.0, 150, 500, 1500 and 5000 μg per plate. No toxicity was observed. Precipitate was observed beginning at 1500 μg per plate with all conditions.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies and the activity of the S9 fraction were found to be satisfactory.

 

No significant increase in the frequency of revertant colonies was recorded for any of the bacterial strains with any dose of the test material, either with or without metabolic activation.

 

These results indicate the test item was negative for the ability to induce reverse mutations at selected loci of several strains of Salmonella typhimurium and at the tryptophan locus of Escherichia coli strain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 August 2017 to 29 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

-Storage conditions: Room temperature, protected from light

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based upon premliminary toxicity results, the doses chosen for the micronucleus assay ranged from 5 to 150 µg/mL for the non activated 4 and 24-hour exposure groups and from 20 to 225 µg/mL for the S9-activated 4-hour exposure group.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Evaluation criteria:

The test substance was considered to have induced a positive response if
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and

• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.

Statistics:

Statistical analysis was performed using the Fisher's exact test (p  0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

observed in preliminary tests, dose selection was adjusted and cytotoxicity was not observed in the definitive assay.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

These results indicate the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Executive summary:

The test material as tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9. Dimethyl sulfoxide (DMSO)was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay. Cytotoxicity [>=50%cytokinesis-blocked proliferation index(CBPI) relative to the vehicle control] was observed at doses >= 200 µg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses >= 600 µg/mL in the non-activated 4 and 24-hour exposure groups and at 2000 µg/mL in the S9-activated 4-hour exposure group. Based upon these results, the doses chosen for the micronucleus assay ranged from 5 to 150 µg/mL for the non‑activated 4 and 24-hour exposure groups and from 20 to 225 µg/mL for the S9-activated 4 -hour exposure group.

In the micronucleus assay, cytotoxicity (>= 50% CBPI relative to the vehicle control) was observed at doses >= 100 µg/mL in the non‑activated 4-hour exposure group; at doses >= 150 µg/mL in the S9‑activated 4-hour exposure group; and at doses > = 75 µg/mL in the non‑activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 25, 50, and 100 µg/mL for the non-activated 4-hour exposure group; 50, 100, and 150 µg/mL for the S9-activated 4-hour exposure group; and 5, 50, and 75 µg/mL for the non‑activated 24-hour exposure group. 

No significant or dose‑dependent increases in micronuclei induction were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2017 to 24 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

-Storage conditions: Room temperature, protected from light

Target gene:

TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay.

Based upon the preliminary results, the concentrations chosen for the definitive mutagenicity assay were 6.25, 12.5, 25, 50, 65, 75 and 100 µg/mL (4-hour treatment with S9), 6.25, 12.5, 25, 50, 100, 120 and 150 µg/mL (4-hour treatment without S9) and 6.25, 12.5, 25, 50, 75, 100 and 125 µg/mL (24-hour treatment without S9).

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

methylmethanesulfonate

Details on test system and experimental conditions:

Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Evaluation criteria:

The average spontaneous mutant frequency of the vehicle control cultures must be within 35 to 140 TFT-resistant mutants/106 surviving cells. Low spontaneous mutant frequencies, i.e., 20 to 34 mutants/106 surviving cells, were considered acceptable if small colony recovery was demonstrated.

The mutant frequency for at least one dose of each positive control must meet the criteria for a positive response.

Cultures treated with a minimum of four concentrations of test substance must be evaluated and their mutant frequencies reported. Results may be accepted, with justification, when only three concentrations of test substance are evaluated and otherwise meet the other criteria for a valid test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the conditions of the assay the test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.

Executive summary:

The test material was evaluated for itsability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cellsin the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at concentrations ≥250 µg/mL at the beginning and end of treatment. Relative suspension growth (RSG) was 10, 49 and 30% at concentrations of 62.5 µg/mL (4-hour treatment with S9), 62.5 µg/mL (4-hour treatment without S9) and 62.5 µg/mL (24-hour treatment without S9), respectively. RSG was or approximated 0% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 6.25, 12.5, 25, 50, 65, 75 and 100 µg/mL (4-hour treatment with S9), 6.25, 12.5, 25, 50, 100, 120 and 150 µg/mL (4-hour treatment without S9) and 6.25, 12.5, 25, 50, 75, 100 and 125 µg/mL (24-hour treatment without S9).

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. Cultures treated at concentrations of 6.25, 12.5, 25, 50 and 65µg/mL (4-hour treatment with S9), 12.5, 25, 50, 100 and 120 µg/mL (4-hour treatment without S9) and 6.25, 12.5, 25, 50 and 75 µg/mL (24-hour treatment without S9) exhibited 16 to 97%, 17 to 112% and 13 to 105% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 16 to 110% (4‑hour treatment with S9), 17 to 106% (4-hour treatment without S9) and 11 to 113% (24‑hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10⁶clonable cells were observed under any treatment condition. 

These results indicate the test material was negative for the ability to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key study:

In an OECD Guideline 471 Study (in vitro gene mutation study in bacteria), conducted according to GLP,Butanedioic acid, 2-[(dipropoxyphosphinothioyl)thio]-, 1,4-dibutyl ester was considered to be non-mutagenic under the conditions of this test (SafePharm Laboratories, 1997).

In an OECD Guideline 487, the test material was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

In an OECD Guideline 490, the test material was concluded to be negative for the induction of forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro L5178Y/TK+/- mouse lymphoma assay.