O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 Nov - 07 Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

EC Number:

227-376-0

EC Name:

O,O-bis(2-ethylhexyl) hydrogen dithiophosphate

Cas Number:

5810-88-8

Molecular formula:

C16H35O2PS2

IUPAC Name:

O,O-bis(2-ethylhexyl) sulfanylphosphonothioate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/β-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with and without metabolic activation
Main Experiment:
Experiment I:
The pre-experiment served as Experiment I.
Experiment II:
3, 10, 33, 100, 333, 1000 and 2500 μg/plate without metabolic activation
10, 33, 100, 333, 1000, 2500 and 5000 μg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: -S9: 4-nitro-o-phenylene-diamine (10 and 50 µg/plate) for TA98 and TA1537, respectively; +S9: 2-aminoanthracene (2.5 or 10.0 µg/plate) for all strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: Plate incorporation (Experiment 1) and preincubation (Experiment 2)

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: A reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn were used to determine the toxicity of the test substance.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

The means from the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 2500 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation; Experiment 2: at 2500 µg/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 1000 µg/plate without metabolic activation and ≥ 2500 µg/plate with metabolic activation; Experiment 2: ≥ 1000 µg/plate without metabolic activation and at 5000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 1000 µg/plate with and without metabolic activation; Experiment 2: ≥ 1000 µg/plate without metabolic activation and ≥ 2500 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1 and 2: ≥ 333 µg/plate without metabolic activation and ≥ 1000 µg/plate with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 1000 µg/plate without metabolic activation and ≥ 2500 µg/plate with metabolic activation; Experiment 2: ≥ 2500 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 1000 to 5000 μg/plate with S9 mix in both experiments. On the plates precipitation of the test item was observed from 2500 to 5000 μg/plate without S9 mix in experiment I and from 1000 to 5000 μg/plate with S9 mix in both experiments. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

HISTORICAL CONTROL DATA
The data of the positive, solvent and negative (untreated) control fall within the range of the historical control data.
- Ranges of positive historical control data (revertants/plate):
Without S9 mix: TA1535 (334 - 1816); TA1537 (43 - 157); TA98 (211 - 627); TA100 (498 - 2767); WP2uvrA (319 - 4732)
With S9 mix: TA1535 (176 - 668); TA1537 (83 - 434); TA98 (360 - 6586); TA100 (536 - 6076); WP2uvrA (167 - 1265)
- Ranges of negative (solvent/vehicle) historical control data (revertants/plate):
Without S9 mix: TA1535 (6 - 25); TA1537 (6 - 19); TA98 (13 - 43); TA100 (78 - 209); WP2uvrA (27 - 63)
With S9 mix: TA1535 (7 - 26); TA1537 (7 - 30); TA98 (15 - 58); TA100 (73 - 208); WP2uvrA (28 - 72)
- Ranges of negative (untreated) historical control data (revertants/plate):
Without S9 mix: TA1535 (6 - 28); TA1537 (5 - 21); TA98 (12 - 43); TA100 (79 - 217); WP2uvrA (30 - 63)
With S9 mix: TA1535 (7 - 26); TA1537 (7 - 31); TA98 (11 - 57); TA100 (85 - 218); WP2uvrA (36 - 88)

Any other information on results incl. tables

Table 1: Summary of Results of Experiment 1

With or without S9-Mix	Test substance concentration (μg/plate)	Average number of colonies of 3 plates±S.D.
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Solvent control (DMF)	179± 33	13± 3	36± 7	25 ± 7	11 ± 5
	Untreated	196± 11	11± 2	38± 11	30 ± 5	10 ± 1
–	3	176 ± 13	12 ± 6	37 ± 6	26 ± 10	10 ± 2
–	10	188 ± 5	10 ± 1	44 ± 3	23 ± 6	14 ± 3
–	33	189 ± 9	10 ± 1	44 ± 7	19 ± 3	12 ± 5
–	100	137 ± 10	14 ± 3	39 ± 3	20 ± 1	12 ± 4
–	333	42 ± 3	9 ± 4	23 ± 4	18 ± 6	9 ± 3
–	1000	6 ± 1	7 ± 2	13 ± 2	11 ± 5	4 ± 2
	2500	3 ± 1 PM	2 ± 0 P	4 ± 2 P	2 ± 1 PM	3 ± 1 P
–	5000	1 ± 1 PM	1 ± 1 PM	1 ± 1 PM	1 ± 1 PM	1 ± 1 PM
Positive controls, –S9	Name	SA	SA	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10.0	10.0	2.0 µL	10.0	50.0
	Average number of colonies of 3 plates ± S.D.	2453 ± 232	1559 ± 21	1098 ± 51	472 ± 28	88 ± 9
+	Solvent control (DMF)	164± 13	9± 2	46± 8	34 ± 9	12 ± 3
	Untreated	209± 8	13± 5	49± 6	40 ± 2	18 ± 3
+	3	166 ± 10	10 ± 1	52 ± 6	45 ± 7	15 ± 5
+	10	181 ± 11	12 ± 3	55 ± 6	32 ± 5	12 ± 3
+	33	179 ± 5	13 ± 2	42 ± 6	33 ± 7	14 ± 0
+	100	179 ± 27	10 ± 4	56 ± 6	29 ± 2	8 ± 2
+	333	127 ± 3	13 ± 4	37 ± 4	31 ± 6	10 ± 5
+	1000	33 ± 13 P	11 ± 1 P	29 ± 5 P	14 ± 4 PM	9 ± 4 P
	2500	9 ± 2 PM	8 ± 2 PM	15 ± 2 PM	6 ± 2 PM	3 ± 2 PM
+	5000	2 ± 1 PM	3 ± 1 PM	2 ± 1 PM	1 ± 1 PM	1 ± 1 PM
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	10.0	2.5	2.5
	Average number of colonies of 3 plates ± S.D.	5504 ± 176	447 ± 46	484 ± 50	5015 ± 542	228 ± 36

MMS = Methyl methane sulfonate

SA = Sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

2AA = 2-Aminoanthracene

P = Precipitate

M = Manual count

Table 2: Summary of Results of Experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Average number of colonies of 3 plates±S.D.
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Solvent control (DMF)	121± 9	11± 3	38± 7	26 ± 6	10 ± 4
	Untreated	169± 5	11± 3	38± 2	26 ± 5	11 ± 7
–	3	111 ± 11	15 ± 5	33 ± 8	32 ± 2	10 ± 4
–	10	118 ± 14	12 ± 3	41 ± 1	23 ± 7	8 ± 2
–	33	118 ± 13	14 ± 2	42 ± 5	23 ± 8	12 ± 3
–	100	95 ± 15	11 ± 1	43 ± 3	27 ± 3	11 ± 2
–	333	20 ± 1	11 ± 4	40 ± 14	16 ± 6	9 ± 3
–	1000	5 ± 1	6 ± 1 R	27 ± 4	3 ± 1	6 ± 1 R
	2500	1 ± 1	5 ± 2 MR	19 ± 6 R	0 ± 0	5 ± 1 MR
Positive controls, –S9	Name	SA	SA	MMS	4-NOPD	4-NOPD
	Concentrations (μg/plate)	10.0	10.0	2.0 µL	10.0	50.0
	Average number of colonies of 3 plates ± S.D.	2277 ± 182	1509 ± 101	654 ± 71	466 ± 13	66 ± 9
+	Solvent control (DMF)	125± 13	12± 2	42± 10	32 ± 4	11 ± 4
	Untreated	176± 2	11± 5	51± 2	36 ± 4	12 ± 1
+	10	120 ± 13	12 ± 5	46 ± 5	31 ± 10	10 ± 5
+	33	114 ± 11	11 ± 2	50 ± 8	26 ± 2	10 ± 4
+	100	120 ± 10	15 ± 4	49 ± 12	29 ± 3	9 ± 4
+	333	70 ± 6	13 ± 4	35 ± 7	29 ± 8	8 ± 3
+	1000	49 ± 15 P	11 ± 1 P	21 ± 6 PM	19 ± 4 P	7 ± 3 P
+	2500	16 ± 5 PM	11 ± 2 PM	12 ± 1 PM	13 ± 2 PM	10 ± 8 PM
	5000	1 ± 1 PM	10 ± 2 PM	10 ± 3 PM	2 ± 1 PM	3 ± 1 PM
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2.5	2.5	10.0	2.5	2.5
	Average number of colonies of 3 plates ± S.D.	3747 ± 355	406 ± 33	329 ± 26	4353 ± 1091	106 ± 19

MMS = Methyl methane sulfonate

SA = Sodium azide

4-NOPD = 4-nitro-o-phenylene-diamine

2AA = 2-Aminoanthracene

P = Precipitate

M = Manual count

R = Reduced background growth