Diphenyl phosphorochloridate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

BACTERIAL REVERSE MUTATION ASSAY (AMES TEST) Negative with and without metabolic activation (S9), (S. typhimurium, TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA), OECD 471, Wagner & Hines (2008)

Link to relevant study records

Reference

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:

05 June 2008 to 25 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine requirement in the Salmonella typhimurium strains, tryptophan requirement in the Escherichia coli strain.

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

- Preliminary toxicity assay: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333 and 5000 µg/plate
- Mutagenicity assay: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: The test material formed a soluble and clear solution in DMF at 500 mg/mL, the maximum concentration tested.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Minimal top agar containing 0.8 % (w/v) agar and 0.5 % (w/v) NaCl was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water per 100 mL. Bottom agar (Vogel-Bonner minimal medium E) contained 1.5 % (w/v) agar supplemented with 2.5 % (w/v) Oxoid nutrient Broth No. 2
0.5 mL of S9 or Sham mix, 100 µL of tester strain (10⁹ cells/mL) and 50 µL of vehicle, test material dilution or positive control were added to 2.0 mL of selective top agar at 45 ± 2 °C.
The mixture was vortexed and overlaid onto 25 mL minimal bottom agar. After setting, the plates were inverted and incubated at 37 ± 2 °C.

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS: 3 in the mutagenicity assay

DETERMINATION OF CYTOTOXICITY
- Method: Condition of the background lawn

Evaluation criteria:

Positive response: A dose-related increase in the mean number of revertants per plate of at least one tester strain over a minimum of two increasing test concentrations. TA 1535 and TA 1537 were considered positive if a three-fold increase was observed at the peak of the dose-response. For all other tester strains, the required increase was two-fold.
Equivocal response: A biologically relevant increase in revertant count that partially meets only one of the conditions for a positive response (a dose response relationship or an increase above the stated thresholds for the tester strains).
Negative response: A response that meets neither the criteria for a positive response nor an equivocal response.

Species / strain:

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

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:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation occurred during the study
- Other confounding effects: Due to contaminants in the bottom agar in the mutagenicity assay for the TA 100 plates with S9 and also the positive control plates for strain TA 1535 with S9, revertant colonies were counted by hand rather than using an automated colony counter. Acceptable vehicle and positive control values showed that the test system was functioning correctly despite this observation.

RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was observed in the screening test.

COMPARISON WITH HISTORICAL CONTROL DATA: The control data were comparable to the historical control data for the performing laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Summary of the Mutagenicity Assay Results in the Absence of Exogenous Metabolic Activation (S9)

Dose (µg/plate)	Average Number of Revertants Per Plate ± Standard Deviation
	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle control	11 ± 4	132 ± 24	6 ± 1	10 ± 3	26 ± 4
50	10 ± 6	119 ± 4	6 ± 3	10 ± 1	19 ± 5
150	9 ± 2	121 ± 5	8 ± 1	7 ± 4	15 ± 3
500	7 ± 5	115 ± 14	9 ± 4	7 ± 3	20 ± 5
1500	8 ± 4	106 ± 8	8 ± 2	7 ± 1	20 ± 3
5000	9 ± 2	119 ± 13	12 ± 3	8 ± 2	26 ± 8
Positive control*	107 ± 10	469 ± 38	271 ± 37	1304 ± 80	160 ± 22

 *Positive controls: TA 98 2-nitrofluorene at 1.0 µg/plate; TA 100 and TA 1535 sodium azide 1.0 µg/plate; TA 1537 9-aminoacridine75 µg/plate; and WP2 uvrA methyl methanesulfonate 1000 µg/plate.

Table 2: Summary of the Mutagenicity Assay Results in the presence of exogenous metabolic activation (S9)

Dose (µg/plate)	Average Number of Revertants Per Plate ± Standard Deviation
	TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Vehicle control	16 ± 2	93 ± 7	6 ± 1	7 ± 3	19 ± 4
50	15 ± 7	91 ± 7	11 ± 2	5 ± 3	17 ± 7
150	17 ± 5	89 ± 18	9 ± 2	5 ± 3	20 ± 6
500	19 ± 2	98 ± 6	9 ± 2	5 ± 3	25 ± 10
1500	15 ± 3	86 ± 12	12 ± 3	6 ± 2	21 ± 8
5000	15 ± 6	102 ± 13	6 ± 2	6 ± 2	22 ± 2
Positive control*	453 ± 126	481 ± 46	61 ± 21	97 ± 6	273 ± 76

*Positive controls: TA 98, TA 100, TA 1535 and TA 1537 2-aminoanthracene at 1.0 µg/plate; and WP2 uvrA 2-aminoanthracene at 10 µg/plate.

Conclusions:

Interpretation of results (migrated information):
negative in the presence and absence of exogenous metabolic activation

Under the conditions of the study, the test material was found to be non-mutagenic in the bacterial reverse mutation assay (Ames test) in the presence and absence of exogenous metabolic activation in the tester strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA.

Executive summary:

The mutagenicity of the test material was evaluated in a bacterial reverse mutation assay (Ames test) conducted in accordance with the standardised guideline OECD 471 under GLP conditions.

The test material was evaluated using the plate incorporation method using tester strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA in the presence and absence of an exogenous metabolic activation system (S9 mix). The assay was performed in two phases; firstly, a preliminary toxicity assay was conducted to establish the dose range and in the second phase the mutagenicity assay was conducted.

The dose levels tested in the preliminary assay were 6.7, 10, 33, 67, 100, 33, 667, 1000, 333 and 5000 µg/plate. On the basis of this, the dose levels selected for the mutagenicity assay were 50, 150, 500, 1500 and 5000 µg/plate.

No cytotoxicity was observed at any dose level with any tester strain both in the cytotoxicity test and the mutagenicity test. The test material formed a clear solution in the chosen vehicle (DMF); no precipitation was observed in any of the tester plates.

No positive or equivocal responses were recorded in any of the tester strains used in the mutagenicity assay.

Under the conditions of the study, the test material was found to be non-mutagenic in the presence and absence of exogenous metabolic activation in the tester strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenicity of the test material was evaluated the key study Wagner & Hines (2008) using a bacterial reverse mutation assay (Ames test) conducted in accordance with the standardised guideline OECD 471 under GLP conditions. The study was assigned a reliability score of 1 in accordance with the criteria for assessing data quality described in Klimisch et al. (1997).

The test material was evaluated using the plate incorporation method using tester strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA in the presence and absence of an exogenous metabolic activation system (S9 mix). The assay was performed in two phases; firstly, a preliminary toxicity assay was conducted to establish the dose range and in the second phase the mutagenicity assay was conducted.

The dose levels tested in the preliminary assay were 6.7, 10, 33, 67, 100, 33, 667, 1000, 333 and 5000 µg/plate. On the basis of this, the dose levels selected for the mutagenicity assay were 50, 150, 500, 1500 and 5000 µg/plate.

No cytotoxicity was observed at any dose level with any tester strain both in the cytotoxicity test and the mutagenicity test. The test material formed a clear solution in the chosen vehicle (DMF); no precipitation was observed in any of the tester plates.

No positive or equivocal responses were recorded in any of the tester strains used in the mutagenicity assay.

Under the conditions of the study, the test material was found to be non-mutagenic in the presence and absence of exogenous metabolic activation in the tester strains S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvrA.

Justification for selection of genetic toxicity endpoint
A single good quality study was available for evaluation.

Justification for classification or non-classification

The available information is not sufficient to draw conclusion on the germ-cell mutagenicity potential of the substance in accordance with Annex I of the regulation EC 1272/2008 (CLP).