Sodium O,O-diisobutyl dithiophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471: negative

OECD 473: negative

OECD 476: negative

Link to relevant study records

Reference

Reference 1

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:

2013

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus. This gene is situated in man, hamster and other species at the X-chromosome. The gene product, hypoxanthine guanine phosphoribosyl transferase, an enzyme which is not vital for the cell, activates the purine analogues 8-azaguanine and 6-thioguanine (used in this assay) to toxic metabolites. Mutants which do not synthesize the active enzyme are resistant to high concentrations of 6-thioguanine or 8-azaguanine. Such mutants can be obtained by several types of mutations since a simple destruction of a gene ('forward mutation') is sufficient.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's modified Eagle-Medium supplemented with 10% fetal calf serum2, penicillin2 (100 U/mL) and streptomycin2 (100 µg/mL) called DMEM-FCS. Cultures are incubated at 37°C in a humidified atmosphere (90%) containing 10% CO2. For subculturing, a trypsin (0.05%)-EDTA (ethylene¬diamine¬tetra¬acetic acid, 0.02%) solution in modified Puck's salt solution A2 is used. Exposure to the test item in the presence of S9 mix is performed in Dulbecco's phosphate buffered saline (PBS)2 which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid)2 pH 7.4 (PBS-HEPES). The cells are periodically checked for the absence of mycoplasma contamination by using the HOECHST stain 33258. The spontaneous mutation rate is continuously monitored.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

a microsomal preparation derived from Aroclor 1254-induced rat liver.

Test concentrations with justification for top dose:

Preliminary study: 10, 25, 100, 250, 1000, 2500 and 5000 μg IBP1-Na/mL
Main study: (4h) 156.3, 312.5, 625, 1250, 2500 μg IBP1-Na/mL. (24h) 78.13, 156.3, 312.5, 625, 1250 μg IBP1-Na/mL.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

Remarks on result:

other: all strains/cell types tested

Conclusions:

Under the present test conditions, tested up to a cytotoxic concentration, caused no mutagenic effect in the Chinese hamster lung fibroblasts (V79) carried out without and with metabolic activation.
Remark: The test solution contains NaOH due to the production method.

Executive summary:

The genetic toxicity HPRT test was performed according to OECD Guideline 476 under GLP compliance. Chinese hamster lung fibroblasts were used for strain. both with and without metabolic activation. No genotoxicity was detected. No cytotoxcity nor precipitates were detected.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-29 - 2012-11-01

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from healthy donors known to be without any medication and collected in heparinised vessels. Small innocula of whole blood (0.5 mL) were added to tubes containing 5 mL of complete culture medium. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254 was prepared according to MARON and AMES (1983). S9 was collected from 20 - 30 rats.

Test concentrations with justification for top dose:

1, 156.3, 312.5, 625, 1250 and 2500 µg IBP1-Na per mL medium.

Vehicle / solvent:

dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

was used as the positive control for the study in the absence of metabolic activation.

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

was used as the positive control for the study in the presence of metabolic activation.

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 hours in experiment 1 and 4 & 24 hours in experiment 2
SPINDLE INHIBITOR (cytogenetic assays): colcemid® to accumulate cells in a metaphase-like stage of mitosis (c-metaphase).

Evaluation criteria:

The test item is judged to have mutagenic properties with respect to chromosomal or chromatid change, if the following criteria are fulfilled:
o the number of chromosomal aberrations is significantly (at p ≤ 0.05) increased compared with the solvent control
o the increase observed is concentration-dependent
o both duplicate cultures lead to similar results
o the increase should not occur in the severely cytotoxic range (mitotic index <0.25), as it is known that high cytotoxicity causes artefacts in the form of aberrations in in vitro chromosomal aberration tests
o a reproducible increase in the number of cells with chromosomal aberrations.

Statistics:

The assessment was carried out by a comparison of the number of chromosome aberrations of the samples with those of the solvent control, using the exact test of R. A. FISHER (p ≤ 0.05) as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society: Report of the UKEMS subcommittee on guidelines for mutagenicity testing, part III, Statistical evaluation of mutagenicity test data, 1989).

Species / strain:

mammalian cell line, other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity was noted in the experiment without and with metabolic activation (24-h and 4-h exposure) at 2500 µg IBP1-Na /mL. Haemolysis was noted at the concentration of 2500 µg/mL in both experiments.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: human peripheral lymphocytes

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, tested up to cytotoxic concentrations, in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.
Remark: the test solution contains NaOH due to the production method.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Executive summary:

The test was performed according to the OECD guideline 473 under GLP compliance. Human peripheral lymphocytes was used for test. Metabolic activation was used both with and without. Blank, vehicle control and positive control was tested. No genotoxicity was detected, cytotoxicity was noted in the experiment without and with metabolic activation (24-h and 4-h exposure) at 2500 µg test item/mL. Haemolysis was noted at the concentration of 2500 µg/mL in both experiments.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Strain Designation Histidine Gene Locus Affected Additional Mutations Type of Mutation Detected
Repair LPS Plasmids
TA 98#1 his D 3052 uvr B- rfa- pKM 101 Frameshift
TA 100#1 his G 46 uvr B- rfa- pKM 101 Base-pair substitution
TA 102#1 his G 428 wild-type rfa- pKM 101 / pAQ1 Base-pair substitution
TA 1535#2 his G 46 uvr B- rfa- none Base-pair substitution
TA 1537#2 his C 3076 uvr B- rfa- none Frameshift


rfa-: partial loss of lipopolysaccharide (LPS) barrier that causes increased permeability to macromolecules
uvr B-: loss of DNA excision repair system
pKM 101: R-factor plasmid, thought to cause an increased error-prone DNA repair
pAQ1: plasmid, carrier of tetracycline resistance
#1 resistance to Ampicillin
#2 non-resistance to Ampicillin

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

a microsomal preparation derived from Aroclor 1254-induced rat liver.

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: aqua ad iniectabilia ( Batch no. 123618002; B. Braun Melsungen AG, 34212 Melsungen, Germany)

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

in aqua ad iniectabilia (10 µg/plate) : TA 1535, TA 100

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

in DMSO (10 µg/plate) : TA 98

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

in ethanol, abs. (100 µg/plate) : TA 1537

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in DMSO (10 µg/plate) : TA 102

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

in DMSO (10 µg/plate) : TA 98, TA 102, TA 1537

Untreated negative controls:

yes

Remarks:

aqua ad iniectabilia

Positive controls:

yes

Positive control substance:

other: 2-amino-anthracene

Remarks:

in DMSO (2 µg/plate) : TA 100, TA 1535

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48-72 hours at 37°C

Evaluation criteria:

a test item is considered to show a positive response if
- the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY, see COLQUHOUN, D.: Lectures on Biostatistics, Clarendon Press, Oxford (1971)) compared with the solvent control to at least 2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent experiments;
- in addition, a significant (p  0.05) concentration (log value)-related effect (Spearman’s rank correlation coefficient, see COLQUHOUN, D.: Lectures on Biostatistics, Clarendon Press, Oxford (1971)) is observed;
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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

Species / strain:

S. typhimurium TA 102

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:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the present test conditions, tested up to a concentration of 5000 µg i-Butyl-dtp-Na/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.
Remark: the test solution contains NaOH due to the production method.

Executive summary:

The test was performed according to the OECD guideline 471 under GLP compliance. S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 strain were used. Metabolic activation was used both with and without. Vehicle control, blank and positive control was tested. No genotoxicity, no cytotoxicity was detected.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

Justification for selection of genetic toxicity endpoint
The results of all three available genetic toxicity studies are negative.

Justification for classification or non-classification

Based on the OECD Guideline studies with the test item no classification for genetic toxicity according to Regulation (EC) No 1272/2008 is warranted for the test item.