Bis(O,O-diisopropyl dithiophosphate)bis(cyclohexylamine) zinc

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Mar - 29 Apr 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

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

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

O,O-bis(propan-2-yl) [({[bis(propan-2-yloxy)(sulfanylidene)-λ⁵-phosphanyl]sulfanyl}bis(cyclohexylazaniumyl)zinciodiuide)sulfanyl]phosphonothioate

EC Number:

809-986-4

Cas Number:

52585-16-7

Molecular formula:

C24H54N2O4P2S4Zn

IUPAC Name:

O,O-bis(propan-2-yl) [({[bis(propan-2-yloxy)(sulfanylidene)-λ⁵-phosphanyl]sulfanyl}bis(cyclohexylazaniumyl)zinciodiuide)sulfanyl]phosphonothioate

Details on test material:

- Analytical purity: 90%
- Batch No.: 0600016235
- Expiry date: 18 Apr 2017
- Appearance: Beige solid
- Storage conditions: At room temperature

Method

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: 1 male donor, aged 21 years (Experiment I); 1 female donor, aged 33 years (Experiment II)
- Whole blood was used
- Methods for maintenance in cell culture: 3 µg/mL phytohemeagglutinine in medium; cells were cultured at 37 °C with 5.5% CO2 in humidified air

MEDIA USED
- Type and identity of media including CO2 concentration: DMEM/F12 (1:1) supplemented with penicillin/streptomycin (100 U/mL and 100 µg/mL, respectively), FBS (10%), HEPES (10 mM) and heparin (125 U.S.P.-U/mL)

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:

Experiment I:
4 h treatment (without metabolic activation): 69.63, 121.9 and 213.2 µg/mL
4 h treatment (with metabolic activation): 39.79, 69.63 and 121.9 µg/mL

Experiment II:
20 h treatment (without metabolic activation): 79.01, 118.5 and 177.8 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin (125 ng/mL in deionized water (- S9 mix, 4 h, continuous treatment))

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation method: 48 h
- Exposure duration: 4 and 20 h
- Fixation time: 40 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: Duplicates each in 2 independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a cleas microscope slide and thereafter stained with Giemsa.

NUMBER OF CELLS EVALUATED: 500 binucleated cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. According to the study director, the criteria of Countryman and Heddle (1976) were taken for the evaluation of micronuclei.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index was determined and cytotoxicity was expressed as %cytostasis.

OTHER EXAMINATIONS:
- Other: Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

[Reference: Countryman P.I. and Heddle J.A. (1976): The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.]

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% confidence interval).

A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% confidence interval).

Statistics:

Statistical significance will be confirmed by the Chi square test (α < 0.05), using the validated R Script CHI2.Rnw for those values that indicate an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I (4 h): ≥ 373.2 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I (4 h): ≥ 373.2 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

lymphocytes: lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Exp. II (20 h): at 177.8 µg/mL

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. II (20 h): ≥ 400.0 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the presence of S9 mix and a 4-h exposure period, a statistical significant increase (1.05%) in micronucleated cells was observed at the lowest evaluated concentration (39.79 μg/mL). In the absence of S9 mix and a 4-h exposure period, a statistical significant increase (1.10%) in micronucleated cells was observed at the highest evaluated concentration (213.2 μg/mL). However, both values can be declared as biologically irrelevant, because they are well within the range of the historical laboratory control data (0.02 – 1.15% in the absence and 0.08 – 1.20% in the presence of S9 mix).

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH determined in the maximum concentration without metabolic activation was within the same range as in the solvent control.
- Effects of osmolality: The osmolality of the solvent control did not differ from the osmolality of the maximum concentration without metabolic activation.
- Precipitation: The test substance precipitated at the end of the treatment at 213.2 µg/mL and above with and without metabolic activation in Experiment I. In Experiment II precipitation occured at 400.0 µg/mL at the end of treatment without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, blood cultures were exposed to the test substance at 12.99, 22.74, 39.79, 69.63, 121.9, 213.2, 373.2, 653.1, 1143 and 2000 µg/mL culture medium with and without metabolic activation for 4 h exposure time. Strong cytotoxic effects were determined at concentrations of 373.2 µg/mL and above with and without metabolic activation. Precipitation was observed in the culture medium at concentrations of 213.2 µg/mL and above with and without metabolic activation. Since the pre-test fulfilled the requirements for cytogenetic evaluation, it was designated Experiment I. The concentration levels for Experiment II were selected based on the results of the range-finding test (Experiment 1).

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: the number of mono-, bi- and multi-nucleated cells were determined.

NUMBER OF CELLS WITH MICRONUCLEI
- Binucleated cells per culture were scored for cytogenetic damage.

HISTORICAL CONTROL DATA
- Positive historical control data: The values of the positive control were within the range of the historical control data [ranges: 2.10 - 6.40 (-S9, continuous treatment); 4.15 - 24.00 (-S9; pulse treatment); 2.25 - 11.30 (+S9, pulse treatment)].
- Negative (solvent/vehicle) historical control data: The values of the negative control were within the range of the historical control data [ranges: 0.05 - 1.43 (-S9, continuous treatment); 0.15 - 1.25 (-S9; pulse treatment); 0.15 - 1.35 (+S9, pulse treatment)].

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1: Cytogenicity in Experiment I and II

	Concentration [µg/mL]	mono- nucleated cells	bi-nucleated cells	multi- nucleated cells	proliferation index CBPI	mono- nucleated cells	bi-nucleated cells	multi- nucleated cells	proliferation index CBPI	Mean proliferation index CBPI	Cytostasis [%] *
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	94	329	77	1.97	107	326	67	1.92	1.94
Positive control (MMC)	1.0	168	304	28	1.72	144	304	52	1.82	1.77	18.6
Test substance	69.63	160	304	36	1.75	83	350	67	1.97	1.86	8.8
Test substance	121.9	203	278	19	1.63	246	236	18	1.54	1.59	37.6
Test substance	213.2 P	190	286	24	1.67	211	272	17	1.61	1.64	32.1
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	146	286	68	1.84	162	263	75	1.83	1.82
Positive control (CPA)	15.0	177	276	47	1.74	201	259	40	1.68	1.53	15.1
Test substance	39.79	106	300	94	1.98	99	307	94	1.99	1.88	NC
Test substance	69.63	117	313	70	1.91	147	282	71	1.85	1.86	NC
Test substance	121.9	167	287	46	1.76	145	295	60	1.79	1.68	4.9
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	113	370	17	1.81	116	354	30	1.83	1.84
Positive control (Demecolcin)	15.0	224	276	0	1.55	247	249	4	1.51	1.71	15.1
Test substance	79.01	97	374	29	1.86	94	366	40	1.89	1.98	NC
Test substance	118.5	108	355	37	1.86	107	355	38	1.86	1.88	NC
Test substance	177.8	178	308	14	1.67	169	316	15	1.69	1.79	4.9

P: Precipitation occurred at the end of treatment

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

NC: Not calculated as the CBPI is equal or higher than the solvent control value

Table 2: Number of micronucleated cells

	Concentration [µg/mL]	binucleated cells with n micronuclei culture 1			sum	binucleated cells with n micronuclei culture 2			sum	sum in 2000 binucleated cells	Micronucleated cells [%] **
		1	2	>2		1	2	>2
Experiment I: 4 h without S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	2	0	0	2	2	0	0	2	4	0.2
Positive control (MMC)	1.0	94	10	3	107	100	8	1	109	216	10.80 S
Test substance	69.63	2	1	0	3	6	0	0	6	9	0.45
Test substance	121.9	1	0	0	1	3	0	0	3	4	0.20
Test substance	213.2 P	12	2	0	14	8	0	0	8	22	1.10 S
Experiment I: 4 h with S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	2	0	0	2	6	0	0	6	8	0.40
Positive control (CPA)	15.0	34	4	0	38	33	3	0	36	74	3.70 S
Test substance	39.79	15	0	0	15	6	0	0	6	21	1.05 S
Test substance	69.63	4	0	1	5	5	0	0	5	10	0.50
Test substance	121.9	3	0	0	3	11	0	0	11	14	0.70
Experiment II: 20 h without S9 mix
Vehicle control (Ethanol)	0.5 % (v/v)	9	0	0	9	5	1	0	6	15	0.75
Positive control (Demecolcin)	15.0	20	4	1	25	27	4	2	33	58	2.90 S
Test substance	79.01	9	1	0	10	9	0	0	9	19	0.95
Test substance	118.5	9	0	0	9	6	1	0	7	16	0.80
Test substance	177.8 #	32	2	1	35	29	1	1	311	66	1.65 S

P: Precipitation occurred at the end of treatment

S: The number of micronucleated cells is statistically significantly higher than corresponding control values

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

Table 3: Historical Control Data

Solvent Control without S9
Micronucleated cells in %
	Pulse treatment (4/40)	Continuous treatment (20/40)
No. of experiments	50*	54**
Mean	0.61	0.55
95 % Ctrl limit	0.02 – 1.15	0.05 – 1.05
1x SD	0.27	0.25
2x SD	0.54	0.50
Min	0.15	0.05
Max	1.25	1.43
Solvent Control with S9
Micronucleated cells in %
	Pulse treatment (4/40)
No. of experiments	67***
Mean	0.64
95 % Ctrl limit	0.08 – 1.20
1x SD	0.28
2x SD	0.56
Min	0.15
Max	1.35

* Aqueous solvents – 23 Experiments; Organic solvents – 27 Experiments
** Aqueous solvents – 24 Experiments; Organic solvents – 30 Experiments

*** Aqueous solvents – 24 Experiments; Organic solvents – 43 Experiments

Applicant's summary and conclusion

Conclusions:

In the in vitro micronucleus test conducted with human lymphocytes according to OECD 487, clastogenicity could be evidenced in the absence of metabolic activation after a 20-h exposure period.