O,O-bis(methylphenyl) hydrogen dithiophosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECD 471, Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation: negative
OECD 473,chromosome aberration in mammalian cells (human peripheral lymphocytes) with and without metabolic activation: negative

OECD 476, HPRT-V79 mammalian cell mutagenicity test with and without metabloic activation: negative

Link to relevant study records

Reference

Reference 1

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:

5.0, 15.8, 50, 158, 500 and 1580 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (Batch no. B1810; Honeywell Specialty Chemicals Seelze GmbH, 30926 Seelze Germany)

- Justification for choice of solvent/vehicle: Cresyl-P1 (DANAFLOAT™ 070) was not soluble in dimethyl sulfoxid (DMSO), water, methanol or acetone.

Untreated negative controls:

yes

Remarks:

ethanol

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:

ethanol

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

in DMSO (10 µg/plate) : TA 98

Untreated negative controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

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

Untreated negative controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

in DMSO (10 µg/plate) : TA 102

Untreated negative controls:

yes

Remarks:

ethanol

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:

ethanol

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 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

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:

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 3250 µg/plate, in any of the 5 test strains in two independent experiments w./wo metabolic activation, respective

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the plate incorporation test and the preincubationcubation test, each carried out without and with metabolic activation cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1580 µg /plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It can be concluded that the test material is negative with and without metabolic activation in all tester strains.

Under the present test conditions Cresyl-P1 tested up to a cytotoxic concentration of 1580 µg Cresyl-dtp/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.

Executive summary:

The registered substance was tested for its mutagenicity in an Ames Plate Incorporation Assay according to OECD Guideline 471 and under GLP in Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 with and without metabolic activation.
Under the present test conditions Cresyl-P1 tested up to a cytotoxic concentration of 1580 µg Cresyl-dtp/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.

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

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Tests without metabolic activation (4- and 24-hour exposure)
31.3, 62.5 or 125 µg Cresyl-P1/mL medium (4-h exposure), in the absence of metabolic activation
15.3, 31.3 or 62.5 µg Cresyl-P1/mL medium (24-h exposure), in the absence of metabolic activation

Test with metabolic activation (4-hour exposure)
62.5, 125 or 250 µg Cresyl-P1/mL medium in the presence of metabolic activation

Vehicle / solvent:

Ethanol (Batch no. B1810; Honeywell Specialty Chemicals Seelze GmbH, 30926 Seelze Germany)
Cresyl-P1 was not soluble in DMSO, water, methanol or acetone.

Untreated negative controls:

yes

Remarks:

Ethanol

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:

Ethanol

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:

METHOD OF APPLICATION: in medium

DURATION

- Exposure duration: 4 hours and 24 in the absence of metabolic activation.4 hours in the presence of metabolic activation.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

The registered substance was found to be negative with metabolic activation and negative without metabolic activation.

Under the present test conditions, Cresyl-P1, 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.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Executive summary:

The registered substance was tested for its potential to induce chromosome aberrations in an Chromosome Aberration study in mammalian cells (human peripheral lymphocytes) according to OECD Guideline 473 and under GLP, with and without metabolic activation.

Under the present test conditions, Cresyl-P1, 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.
In the same test, Mitomycin C and cyclophosphamide induced significant damages, which confirmed the validity of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes

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 express the active enzyme are resistant to high concentrations of 6-thioguanine or 8-azaguanine. Such mutants can be obtained by a forward mutation of the hprt locus.

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 cytotoxicity test
The concentrations to be employed in the main experiment were chosen based on the results of a preliminary cytotoxicity study without and with metabolic activation with concentrations of 3.16, 10.0, 31.6, 100, 316, 1000 and 2000 µg/mL medium. In this preliminary test cytotoxicity in form of decreased relative survival compared to the control was noted starting at concentrations of 31.6 or 316 µg/mL medium in the experiments without and with metabolic activation. Test item precipitation was noted macroscopically starting at a concentration of 100 µg/mL medium in the experiments without and with S9 mix. Complete cytotoxicity was noted at concentrations of 1000 and 2000 µg/mL medium in both experiments (see Table 1). No changes in pH or osmolality were noted in the test cultures compared to the negative control treated with DMSO. For details see the Text table 5-1. Hence, 400 µg Cresyl-P1/mL medium was employed as highest concentration for the genotoxicity tests without and with metabolic activation in the main experiment.
Main study
Concentrations of 25, 50, 100, 200 and 400 µg Cresyl-P1/mL medium were selected for the mutagenicity experiment without and with metabolic activation.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Cytotoxicity. in form of decr. rel. survival comp. to the control was noted at conc. 200&400 µg Cresyl-P1/mL medium + and - metabolic act.(4h.exp). Test item precipitation noted macrs.scop. at conc.100 to 400 µg/mL medium in the exp. - and + S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the present test conditions, Cresyl-P1 tested up to the cytotoxic concentration of 400 µg/mL medium, that led to test item precipitation (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Executive summary:

The substance was tested in an HPRT-V79 mammalian cell mutagenicity test according to OECD Guideline 476 and under GLP, with and without metabolic activation in Chinese hamster lung fibroblasts. Under the present test conditions, Cresyl-P1 tested up to the cytotoxic concentration of 400 µg/mL medium, that led to test item precipitation (4-hour exposure, without and with metabolic activation) was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the negative results three valid in vitro tests with the test substance, the substance does not need to be classified according to Regulation (EC) No 1272/2008.