Dibutyl [[bis[(2-ethylhexyl)oxy]phosphinothioyl]thio]succinate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation test

Key study:

In a study performed to the standardised guideline OECD 471, under GLP conditions, the test item has been determined to be not-mutagenic (Safepharm Laboratories Limited, 1997).

 

In vitro micronucleus test

Key study:

In a study performed to the standardised guideline OCED 487, under GLP conditions, test item was determined to be negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

 

In vitro gene mutation in mammalian cells test

Key study:

In a study performed to the standardised guideline OECD 490, under GLP conditions, the test item was determined to be 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.

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:

10 December 1996 to 21 May 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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: >94%
Description: Extremely pale straw coloured liquid

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Obtained from the University of California at Berkeley.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Obtained from the British Industrial Biological Research Association.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500 and 5000 µg/plate.
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.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

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

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
S9 was prepared in-house from the livers of male Sprague-Dawley rats, treated with Aroclor 1254. Prior to use, all batches of S9 were checked for suitability using a recognised mutagenic compound (2AA) and stored at -196 °C in a liquid nitrogen freezer.

S9-Mix and Agar
The S9-mix was prepared at 4 °C as follows:
S9: 5.0 mL
1.65 M KCI/0.4 M MgCI2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A preliminary test using the test material at 0, 50, 150, 500, 1500 and 5000 µg/plate was carried out initially to determine the toxicity of the test material to the tester organisms.

Five concentrations of the test material at 50, 150, 500, 1500 and 500 µg/plate were assayed in triplicate in each tester strain, using the direct plate incorporation method.

All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

A second experiment was performed using methodology as described for experiment 1, using fresh bacterial cultures, test material and control solutions in triplicate.

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically significant increase in mutation rate of at least twice the spontaneous reversion rate in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than two fold at each dose level employed.

Statistics:

A statistical analysis of the data was not required to determine the result of the test.

Key result

Species / strain:

other: TA 100, TA 1535, TA 98, TA 1537 and WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

In a study performed to the standardised guideline OECD 471, under GLP conditions, Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA 100 and Escherichia coli strain WP2uvrA were treated with the test material using the plate incorporation method at five dose levels, in triplicate, both with and without the addition of S9.

 

The vehicle (acetone) control plates produced counts of revertant colonies within the normal range.

 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system.

 

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. The test material has been determined to be not-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 October 2017 to ****

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:

CAS No.: 68413-48-9
Purity: 84%
Description: Clear colorless liquid

Target gene:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y/TK+/- cells, clone 3.7.2C, obtained from the American Type Culture Collection (repository number CRL-9518), Manassas, VA. Each batch of frozen cells was tested and found to be free of mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

S9

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. Visible precipitate was observed at concentrations ≥250 µg/mL at the beginning of treatment and at concentrations ≥500 µg/mL by the end of treatment. Relative suspension growth (RSG) was 10, 42 and 15% at concentrations of 500 µg/mL (4-hour treatment with S9), 500 µg/mL (4-hour treatment without S9) and 125 µg/mL (24-hour treatment without S9), respectively. RSG was 0 to 8% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the initial definitive mutagenicity assay were 15.6, 31.3, 65.2, 125, 250 and 375 µg/mL (4-hour treatment with S9), 31.3, 65.2, 125, 250 and 500 µg/mL (4-hour treatment without S9) and 31.3, 65.2, 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:

Solubility Determination
DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL.

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 lot of S9 was assayed for sterility and its ability to metabolize at least two pro-mutagens to forms mutagenic to Salmonella typhimurium TA100.
The S9 mix was prepared on the day of use and final concentrations of its components in the test system were: DL-isocitric acid (17.4 mM), NADP (sodium salt; 3.0 mM), and S9 (10 µL/mL).

Preliminary Toxicity Test for Selection of Dose Levels
L5178Y/TK+/- cells were exposed to the vehicle alone in duplicate cultures and nine concentrations of test substance using single cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. The maximum concentration evaluated was based on solubility limitations of the test substance in the vehicle; precipitation of the test substance in the treatment medium was determined with the unaided eye at the beginning and end of treatment. The pH of the treatment medium was measured and adjusted if necessary to maintain neutral pH. Osmolality in treatment medium of the vehicle control, the highest concentration, the lowest precipitating concentration and the highest soluble concentration also was measured. Dose levels for the definitive assay were based on post-treatment cytotoxicity (growth inhibition relative to the vehicle control) or solubility limitations of the test substance in the treatment medium.
For the preliminary toxicity assay only, after a 4-hour treatment in the presence and absence of S9, cells were washed with culture medium and cultured in suspension for two days post treatment, with cell concentration adjustment on the first day. After a 24 hour treatment in the absence of S9, cells were washed with culture medium and immediately readjusted to 3 x 10^5 cells/mL. Cells were then cultured in suspension for an additional two days post-treatment with cell concentration adjustment on the first day.

Mouse Lymphoma Assays
L5178Y/TK+/- cells were exposed to the vehicle alone and five to seven concentrations of test substance at appropriate dose intervals using duplicate cultures for 4 hours in the presence and absence of S9, and for 24 hours in the absence of S9. Precipitation was determined with the unaided eye at the beginning and end of treatment.

Treatment of Target Cells
The preparation and addition of the test substance to the test system was carried out under filtered lighting. A mixture containing 50 µL of test substance or vehicle control formulation or 100 µL of positive control formulation were added to treatment medium. All pH adjustments were performed prior to adding S9 or target cells. Either medium or S9 mix (as appropriate) and 6 x 10^6 L5178Y/TK+/- cells were added, cultures were capped tightly, and incubated with mechanical mixing at 37 ± 1 °C for 4 or 24 hours.
For the definitive assay only, at the end of the exposure period, the cells were washed with culture medium, resuspended in F10P, and incubated at 37 ± 1 °C for two days following treatment. Cell population adjustments to 3 x 10^5 cells/mL were made as follows:
• 4 hour treatment – 1 and 2 days after treatment.
• 24 hour treatment – immediately after test substance removal; 2 and 3 days after treatment.
Selection of Mutant Phenotype
Cells from selected dose levels were cultured in triplicate with 2-4 μg TFT/mL at a density of 1 x 10^6 cells/100 mm plate in cloning medium containing 0.22 to 0.24% agar. For estimation of cloning efficiency at the time of selection, 200 cells/100 mm plate were cultured in triplicate in cloning medium without TFT (viable cell (VC) plate). Cultures were incubated under standard conditions (37 ± 1 °C in a humidified atmosphere of 5 ± 1% CO2 in air) for 10 or 11 days. The plates were stored under refrigerated conditions (2-8°C) for 8 days prior to scoring.

The total number of colonies per culture was determined for the VC plates and the total relative growth calculated. The total number of colonies per TFT plate was then determined for those cultures with ≥10% total growth (including at least one concentration between 10 and 20% total growth, if possible). Colonies were counted and the diameter of the TFT colonies from the positive control and vehicle control cultures were determined over a range from 0.2 to 1.1 mm.

Extended Treatment and/or Confirmatory Assay
Verification of a clear positive response was not required (OECD Guideline 490). For negative results without activation, an extended treatment assay was performed in which cultures were continuously exposed to the test substance for 24 hours without S9 activation. The extended treatment assay was performed concurrently with the initial assay. For negative results with S9 activation, a confirmatory assay was not required unless the test substance was known to have specific requirements of metabolism.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Visible precipitate was observed at concentrations ≥375 µg/mL at the beginning and by the end of treatment (4 hour treatment with S9), and at the beginning and by the end of treatment at a concentration of 500 µg/mL (4-hour treatment without S9). The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

Cultures treated at concentrations of 31.3, 65.2, 125, 250 and 375 µg/mL (4-hour treatment with S9) and 31.3, 65.2, 125, 250 and 500 µg/mL (4-hour treatment without S9) exhibited 24 to 84% and 44 to 107% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 24 to 99% (4 hour treatment with S9) and 37 to 116% (4-hour treatment without S9). The initial 24-hour treatment without S9 exhibited insufficient toxicity and was repeated with concentrations 0.5, 5, 25, 50, 100, 200, 300, 500, 750 and 1000 µg/mL. No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under 4 hour treatment with and without S9 treatment conditions.

In the retest of definitive mutagenicity assay (24-hour treatment without S9), visible precipitate was observed at concentrations ≥300 µg/mL at the beginning and end of treatment.
The test substance did not have an adverse impact on the pH of the cultures (pH 7.5 at the top dose).

Cultures treated at concentrations of 0.5, 5, 25, 50 and 100 µg/mL (24-hour treatment without S9) exhibited 98 to 109% RSG, and were cloned. One plate at 0.5µg/mL was lost due to dilution error. Relative total growth of the cloned cultures ranged from 74 to 135% (24 hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed.

Conclusions:

The test substance was determined to be 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.

Executive summary:

In a study performed to the standardised guideline OECD 490, under GLP conditions, the test substance was evaluated for its 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. 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 of treatment and at concentrations ≥500 µg/mL by the end of treatment. Relative suspension growth (RSG) was 10, 42 and 15% at concentrations of 500 µg/mL (4-hour treatment with S9), 500 µg/mL (4-hour treatment without S9) and 125 µg/mL (24-hour treatment without S9), respectively. RSG was 0 to 8% at all higher concentrations using all treatment conditions. Based upon these results, the concentrations chosen for the initial definitive mutagenicity assay were 15.6, 31.3, 65.2, 125, 250 and 375 µg/mL (4-hour treatment with S9), 31.3, 65.2, 125, 250 and 500 µg/mL (4-hour treatment without S9) and 31.3, 65.2, 75, 100 and 125 µg/mL (24-hour treatment without S9).

 

In the initial definitive mutagenicity assay, visible precipitate was observed at concentrations ≥375 µg/mL at the beginning and by the end of treatment (4 hour treatment with S9) and at the beginning and by the end of treatment and at a concentration of 500 µg/mL (4-hour treatment without S9). Cultures treated at concentrations of 31.3, 65.2, 125, 250 and 375 µg/mL (4-hour treatment with S9) and 31.3, 65.2, 125, 250 and 500 µg/mL (4-hour treatment without S9) exhibited 24 to 84% and 44 to 107% RSG, respectively, and were cloned. Relative total growth of the cloned cultures ranged from 24 to 99% (4 hour treatment with S9) and 37 to 116% (4-hour treatment without S9). The initial 24-hour treatment without S9 exhibited insufficient toxicity and was repeated with concentrations 0.5, 5, 25, 50, 100, 200, 300, 500, 750 and 1000 µg/mL. No increases in induced mutant frequency ≥90 mutants/10^6 clonable cells were observed under 4 hour treatment with and without S9 treatment conditions.   

 

In the retest of definitive mutagenicity assay (24-hour treatment without S9), visible precipitate was observed at concentrations ≥300 µg/mL at the beginning and end of treatment. Cultures treated at concentrations of 0.5, 5, 25, 50 and 100 µg/mL exhibited 98 to 109% RSG, and were cloned. One plate at 0.5µg/mL was lost due to dilution error. Relative total growth of the cloned cultures ranged from 74 to 135% (24 hour treatment without S9). No increases in induced mutant frequency ≥90 mutants/106 clonable cells were observed. 

 

The test substance was determined to be 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.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 August 2017 to ****

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:

CAS RN: 68413-48-9
Purity: 84%
Description: Clear colorless liquid

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking, 27 year old female and were cultured in complete medium (RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin) by adding 0.5 mL heparinized blood to a centrifuge tube containing 5 mL of complete medium with 2% phytohemagglutinin. The cultures were incubated under standard conditions (37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air) for 44-48 hours.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Based on the results of the preliminary toxicity test, the doses selected for testing in the micronucleus assay were as follows:
- Non-activated: 1, 5, 50, 100, 200 µg/mL, for 4 and 24 hrs, and
- S9-activated: 300, 600, 700, 1000, 1300, 1500, 1800 µg/mL, for 4 hrs.

Precipitation of the test substance dosing solution in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The highest dose evaluated for the micronuclei was selected based on the following:
- 4-hour (-S9): Visible precipitate at the end of treatment period;
- 4-hour (+S9): 49% cytotoxicity (CBPI relative to the vehicle control), and
- 24-hour (-S9): Visible precipitate at the end of treatment period.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Vinblastine

Details on test system and experimental conditions:

Exogenous Metabolic Activation System
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 final concentration of its components in serum-free RPMI-1640 medium were: S9 (20 µL/mL), MgCl2 (2 mM), KCl (6 mM), glucose-6-phosphate (1 mM), and NADP (sodium salt; 1 mM).

Preliminary Toxicity Test for Selection of Dose Levels
Lymphocytes were exposed to vehicle alone and to nine concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using single cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment. The osmolality in treatment medium of the vehicle, the highest dose, lowest precipitating dose, and the highest soluble dose was measured. The pH of the highest dose in the treatment medium was measured using test tape. Dose levels for the micronucleus assay were based upon post-treatment toxicity (cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control) or visible precipitate in the treatment medium at the conclusion of the treatment period. .After the 4 hour treatment in the non-activated and the S9-activated studies, the cells were centrifuged, the treatment medium was aspirated, the cells were washed with calcium and magnesium free phosphate buffered saline (CMF-PBS), re-fed with complete medium containing cytochalasin B at 6.0 µg/mL and returned to the incubator under standard conditions. For the 24 hour treatment in the non-activated study, cytochalasin B (6.0 µg/mL) was added at the beginning of the treatment.

Micronucleus Assay
Lymphocytes were exposed to vehicle alone and to a minimum of five concentrations of test substance for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 using duplicate cultures. Precipitation of test substance in the treatment medium was determined using unaided eye at the beginning and conclusion of treatment.

Collection of Cells (Preliminary Toxicity Test and Micronucleus Assay)
Cells were collected by centrifugation, swollen with 0.075M KCl, washed with fixative (methanol: glacial acetic acid, 25:1 v/v), and the suspension of fixed cells was applied to glass microscope slides and air-dried. The slides were stained with acridine orange.

Cell Cycle Kinetics Scoring (Preliminary Toxicity Test and Micronucleus Assay)
For the preliminary toxicity test, at least 500 cells, if possible, were evaluated to determine the CBPI at each dose level and the control. For the micronucleus assay, at least 1,000 cells (500 cells per culture), if possible, were evaluated to determine the CBPI at each dose level and the control. The CBPI was determined using the following formula:
CBPI = 1X Mononucleated cells + 2 x Binucleated cells + 3 x Multinucleated cells
Total number of cells scored
% Cytostasis (cytotoxicity) = 100 -100 {(CBPItest-1) /(CBPIvehicle-1)}

Micronucleus Scoring
The slides from at least three test substance concentrations were coded and a minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei. Micronuclei in a binucleated cell (MN-BN) were recorded if they met the following criteria:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.

Evaluation criteria:

The frequency of cells with micronuclei should ideally be within the 95% control limits of the distribution of the historical negative control database. If the concurrent negative control data fall outside the 95% control limits, they may be acceptable as long as these data are not extreme outliers (indicative of experimental or human error). The percentage of micronucleated cells must be significantly greater than the concurrent vehicle control (p ≤ 0.05). In addition, the cytotoxicity response must not exceed the upper limit for the assay (60%). The CBPI of the vehicle control at harvest must be ≥ 1.4. At least 2000 binucleated cells from at least three appropriate test substance concentrations should be analyzable.

The test substance must be tested using a 4-hour treatment with and without S9, as well as a 24 hour treatment without S9. However, all three treatment conditions need not be evaluated in the case of a positive test substance response under any treatment condition.

The maximum concentration evaluated for micronucleus induction must
• produce cytotoxicity in the target range of 55 ± 5%, or
• produce turbidity or a precipitate visible by eye at the end of the treatment with the test substance or
• if no precipitate or limiting cytotoxicity was observed, be 10 mM, 2 mg/mL or 2 µL/mL, whichever is the lowest.

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. The Cochran-Armitage trend test was used to assess dose-responsiveness.

Key result

Species / strain:

lymphocytes: human

Remarks:

4 hour treatment

Metabolic activation:

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

Key result

Species / strain:

lymphocytes: human

Remarks:

4 hour treatment

Metabolic activation:

with

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

Key result

Species / strain:

lymphocytes: human

Remarks:

24 hour treatment

Metabolic activation:

with

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

Conclusions:

The test item was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Executive summary:

The test substance was assessed in a study performed to the standardized guideline OCED 487, under GLP conditions, 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 2000 µg/mL in all three exposure groups. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 200 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 1 to 200 µg/mL for the non activated 4 and 24-hour exposure groups and from 300 to 1800 µg/mL for the S9-activated 4-hour exposure group.

 

In the micronucleus assay, cytotoxicity (≥ 50% CBPI relative to the vehicle control) was not observed at any dose in the non-activated 4 and 24-hour exposure groups. Cytotoxicity was observed at doses ≥ 1000 µg/mL in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥ 100 µg/mL in the non activated 4 and 24-hour exposure groups and at doses ≥ 1000 µg/mL in the S9 activated 4-hour exposure group. The doses selected for evaluation of micronuclei were 1, 50, and 100 µg/mL for the non-activated 4-hour exposure group; 300, 600, and 700 µg/mL for the S9-activated 4-hour exposure group; and 1, 5, and 100 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 item was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available