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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Oct 2019 - 26 May 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.62 (In Vivo Mammalian Alkaline Comet Assay)

Version / remarks:

EC Commission Regulation No. 2017/735

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM

Type of assay:

mammalian comet assay

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

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: Wistar Han

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Age at administration: ca. 54 - 60 days
- Weight at study initiation: 219 - 262 g
- Assigned to test groups randomly: yes
- Housing: in groups; animals were given access to small soft white untreated wood (Aspen) chew blocks and a plastic shelter for environmental enrichment
- Diet: pelleted Envigo Teklad 2014C, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 (minor deviation on one day with 19 °C reported)
- Humidity (%): 40 - 70 (minor deviation noticed on 2 distinct days, with 38% humidity measured for ca. 1 h)
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle/solvent used: corn oil
- Concentration of test material in vehicle: 18.75, 37.5 and 75 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Supplier: KTC Edibles Ltd, United Kingdom
- Lot/batch no.: 9154K

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
An accurately weighed amount of the test item was ground with a mortar and pestle, the vehicle was gradually added during continuous rubbing. The formulations were homogenised for 5 min and sonicated for 5 min until a homogenous suspension was achieved. Formulations were magnetically stirred until and during administration. The test item formulations were prepared and analysed for achieved concentration on each day of dosing.

Duration of treatment / exposure:

2 days

Frequency of treatment:

daily (the second dose being administered approximately 24 hours after the first dose)

Post exposure period:

sampling time point: 3 h after the second dose administration

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

187.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

6 males (test item and vehicle control) and 3 males (positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate (EMS)
- Route of administration: oral gavage (single treatment approximately 3 h prior to termination)
- Doses / concentrations: 200 mg/kg bw / 20 mg/mL (dose volume 10 mL/kg bw)

Examinations

Tissues and cell types examined:

liver, duodenum and glandular stomach

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Dose levels were selected based on the results of a preliminary toxicity in male and female rats (please refer to "Additional information on results"). The highest dose level for the main comet assay was chosen to represent the maximum tolerated dose (MTD), defined as the highest dose that will be tolerated without evidence of study-limiting toxicity, relative to the duration of the study period.

DETAILS OF SINGLE CELL SUSPENSION PREPARATION:
The animals were sacrificed about 3 hours after administration of the second dose. Positive control animals were sacrificed approximately 3 h after a single dosing. Sections of the liver,duodenum and glandular stomach were prepared and placed into ice cold mincing solution; all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using the following tissue specific methods:

Liver
Approximately 0.5 cm³ of liver tissue was cut and washed in fresh complete mincing solution until as much blood was removed as possible. 2 mL of fresh complete mincing solution was added to the liver and then cut into several small pieces. The pieces were transferred to 150 µm bolting cloth held over a falcon tube and the liver was pushed through the cloth. An additional 2 mL of complete mincing solution was added, and any remaining liver pushed through the cloth. The sample was stored on ice until slide preparation.

Duodenum
The duodenum was removed, washed free of waste using complete mincing solution and placed into a falcon tube and covered with approximately 2 mL of mincing solution. The samples were incubated on ice for approximately 30 min (± 10 min). After incubation the tissue was removed and was washed free of waste using complete mincing solution for a second time. The tissue was transferred to a clean Petri dish, 1.5 mL of complete mincing solution was added, and the tissue was minced using a scalpel blade to release the cells. The cells were poured into a clean falcon tube and stored on ice until slide preparation.

Glandular Stomach
The stomach was removed, cut open, washed free from food using mincing solution and incubated in a falcon tube on ice for 30 min (± 10 min). Following incubation, the stomach was removed and surface epithelial was gently scraped using a cell scraper. The layer was discarded, and gastric mucosa was rinsed with fresh mincing solution until all food was removed. 1.5 mL of fresh mincing solution was added to a clean petri dish and the stomach was scraped 4 - 5 times to release the cells. The cells were stored on ice until slide preparation.

DETAILS OF SLIDE PREPARATION:
Comet slides were prepared from all cell suspensions.
Frosted end glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75 µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped. Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8 ºC overnight prior to electrophoresis.
Electrophoresis
The slides were randomly placed onto a dry, level platform of a horizontal electrophoresis unit containing chilled electrophoresis buffer. The slides from each treatment were spread across the platform to avoid any positional effects. The buffer reservoir of the unit was topped up with electrophoresis buffer until the surfaces of the slides are covered. The slides were left for 20 min to allow the DNA to unwind. After alkali unwinding, the slides were electrophoresed at 18 V with a starting current of approx. 300 mA (between 0.7 to 1.0 V/cm) for 30 min.
The temperature of the electrophoresis solution during unwinding and electrophoresis was maintained at a low temperature, usually 2 – 10 °C. When electrophoresis was completed, the slides were washed on three occasions (separated by 5 min intervals) with neutralisation buffer and stored, refrigerated, in lightproof boxes with moistened tissues (to prevent agar dehydration).

METHOD OF ANALYSIS:
Microscopic Examination
Coded slides were stained with SYBR GOLD® (Life Technologies) and visualised under a fluorescence microscope. The comet images from the microscope were captured via a CCD camera and measured using Perceptive Instruments COMET IVTM image analysis system. Initially the slides were examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells (i.e. hedgehog cells). These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.
50 cells were scored per slide to give a total number of 150 cells per tissue per animal. The extent of DNA migration and hence damage is reflected by the percentage of tail intensity (% TI) defined as the fluorescence detected by image analysis in the tail, which is proportional to the amount of DNA that has moved from the head region into the comet tail.

OTHER:
Histopathology
Sections of the liver, duodenum and glandular stomach were stored in 10% buffered formalin. Sections of the duodenum and glandular stomach from the vehicle control animals and animals administered test item doses of 187.5, 375 and 750 mg/kg bw/day were embedded in paraffin wax, sectioned and stained with haematoxylin and eosin and assessed for signs of cytotoxicity, necrosis and apoptosis.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
a) At least one of the test doses exhibits a statistically significant increase in % tail intensity compared with the concurrent vehicle control.
b) The increase in % tail intensity is dose-related when evaluated with an appropriate trend test.
c) Any of the results are outside the distribution of the historical vehicle control data.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if:
a) None of the test concentrations exhibits a statistically significant increase in % tail intensity compared with the concurrent vehicle control.
b) There is no concentration-related increase in % tail intensity when evaluated with an appropriate trend test.
c) All results are inside the distribution of the historical vehicle control data.

There is no requirement for verification of a clearly positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above and in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. dose spacing, other routes of administration, other sampling times or other tissues) could be useful.
In rare cases, even after further investigations, the data set will preclude making a conclusion of positive or negative results, and will therefore be concluded as equivocal.
To assess the biological relevance of a positive or equivocal result, cytotoxicity at the target tissue should also be discussed. Histopathological information can help in the interpretation of a positive result in the comet assay. Careful interpretation of increased or decreased % tail DNA in the presence of severe cytotoxicity is therefore essential.

Statistics:

Please refer to pdf document "Statistical analysis" under "attached background material"

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: liver tissue

Test results 2

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

statistically significant increase in % tail intensity at 187.5, 375 and 750 mg/kg bw/day

Toxicity:

yes

Remarks:

Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: duodenum tissue

Test results 3

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

statistically significant increase in % tail intensity at 750 mg/kg bw/day

Toxicity:

yes

Remarks:

Clinical signs were observed at all dose levels and comprised piloerection at ≥ 187.5 mg/kg bw/day and fast breathing at 750 mg/kg bw/day.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: glandular stomach tissue

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the preliminary toxicity study, the animals were initially treated at 1000 mg/kg bw/day with 2 doses administered 24 h apart. At 1000 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, piloerection, elevated gait and prominent eyes (both). The clinical signs observed were mainly classified as moderate and persisted until the end of the working day. On the basis of this result the maximum tolerated dose had been exceeded, therefore, an additional group of two male and two female animals was administered 500 mg/kg bw/day. At 500 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, piloerection, elevated gait and prominent eyes (both). Clinical signs were mainly classified as mild and any moderate signs transitioned to mild after around 15 minutes post-gavage. On the basis of this result the maximum tolerated dose had not been achieved, therefore, an additional group of two male and two female animals was administered 750 mg/kg bw/day. At 750 mg/kg bw/day, clinical signs of toxicity in male and female animals included fast breathing, salivation, pilo erection, elevated gait and prominent eyes (both). Clinical signs were mainly classified as mild and moderate signs were transient in nature.
On the basis of these results the maximum tolerated dose was considered to be 750 mg/kg bw/day. There was not considered to be a substantial difference in toxicity between the sexes, therefore, in line with current guidelines, the comet test was conducted using male animals only.

RESULTS OF DEFINITIVE STUDY
Hedgehog cells were excluded from the analysis, along with any cells that had unusual staining artefects.

- Results on tail intensity (% TI):

Vehicle control group:
The vehicle control group mean and median % tail intensity values for the liver, duodenum and glandular stomach of male Crl: Wistar Han rats were within the current vehicle historical control range for the individual tissue (95% confidence limits). For details on historical control data please refer to Tables 5 and 6 under "Any other information on results incl. tables".

Positive control group:
The positive control compound, EMS, produced a significant increase (p < 0.001) in the median % TI when compared to vehicle control values in all tissues analysed and all % TI values were comparable to the positive historical control range. For details on historical control data please refer to Tables 5 and 6 under "Any other information on results incl. tables".

Test item group:
Liver:
There were no statistically significant increases observed in the median % TI in the liver of male rats administered 187.5, 375 or 750 mg/kg bw/day. The group (mean and median) % TI values from all treatment groups were within the current vehicle historical control range (95% confidence limits). For details please refer to Table 2 under "Additional information on results incl. tables".

Duodenum:
There were statistically significant increases observed in the median % TI in the duodenum of male rats administered 187.5 mg/kg bw/day (p<0.05), 375 and 750 mg/kg bw/day (p < 0.001). The group (mean and median) % TI values were outside of the current vehicle historical control range (95% confidence limits). For details please refer to Table 3 under "Additional information on results incl. tables".

Glandular stomach:
There were statistically significant increases observed in the median % TI in the glandular stomach of male rats administered 750 mg/kg bw/day (p < 0.001). The group (mean and median) % TI values were outside of the current vehicle historical control range (95% confidence limits). For the 187.5 and 375 mg/kg bw/day dose groups, no statistically significant increases in the median % TI in the glandular stomach were observed and the group (mean and median) %TI values were within the current vehicle historical control range (95% confidence limits). For details please refer to Table 4 under "Additional information on results incl. tables".

- Results on Hedgehog cells:
In the liver, hedgehog cells were observed after administration of 750 mg/kg bw/day only.
In the duodenum, there was a dose-related increase in the number of hedgehog cells observed at 187.5, 375 or 750 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 187.5, 375 or 750 mg/kg bw/day: 0.5, 14.2, 23.7 and 33.2 respectively).
In the glandular stomach, there was a dose-related increase in the number of hedgehog cells observed after administration of 187.5, 375 or 750 mg/kg bw/day, compared to the concurrent vehicle control (group mean: vehicle control, 187.5, 375 or 750 mg/kg bw/day: 0.0, 0.0, 2.8 and 29.3 respectively).

- Results on histopathology:
There were no test item-related changes in the glandular stomach and the duodenum. The liver was not examined for histopathological changes.

Any other information on results incl. tables

Mortality and clinical signs of toxicity:

No mortalities were observed throughout the duration of the comet assay. Clinical signs of toxicity comprised piloerection in 5/6 animals at 187.5 mg/kg bw/day, in 2/6 animals at 375 mg/kg bw/day and in 6/6 animals at 750 mg/kg bw/day. In addition, fast breathing was observed in 1/6 animals at 750 mg/kg bw/day.

Body weight development:

Marginal body weight loss was noted in animals of the 375 mg/kg bw/day group (-0.4%) and in animals of the 750 mg/kg bw/day group (-1.5%) when compared to the vehicle control animals.

Result tables

Table 2: Summary of results in liver tissue

Male animal data - Liver
Treatment	Dose (mg/kg bw/day)	Number of cells scored	Group mean tail intensity% (SD) #	Group mean of median tail intensity% (SD) #
Vehicle	-	900	2.57 (0.5)	0.58 (0.5)
Test item	187.5	900	2.69 (0.6)	0.47 (0.3)
Test item	375	900	3.03 (0.6)	0.50 (0.3)
Test item	750	900	3.01 (0.8)	0.47 (0.4)
EMS	200	450	54.14 (8.4)	55.16 ^L+***(9.5)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Table 3: Summary of results in duodenum tissue

Male animal data - Duodenum
Treatment	Dose (mg/kg bw/day)	Number of cells scored	Group mean tail intensity% (SD) #	Group mean of median tail intensity% (SD) #
Vehicle	-	900	5.83 (1.4)	2.06 (1.0)
Test item	187.5	900	7.65 (1.9)	3.87 *(0.9)
Test item	375	900	10.35 (1.9)	5.38 ***(1.3)
Test item	750	900	13.81 (2.0)	7.26 ***(1.3)
EMS	200	450	61.96 (6.0)	63.66 ^L+***(8.1)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001, * p < 0.05 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Table 4: Summary of results in glandular stomach tissue

Male animal data - Glandular stomach
Treatment	Dose (mg/kg bw/day)	Number of cells scored	Group mean tail intensity% (SD) #	Group mean of median tail intensity% (SD) #
Vehicle	-	900	4.74 (1.1)	1.69 (0.9)
Test item	187.5	900	3.39 (0.5)	0.93 ^L(0.3)
Test item	375	900	4.98 (2.4)	2.00 ^L(1.3)
Test item	750	900	17.36 (2.7)	11.42 ^L***(2.7)
EMS	200	450	58.11 (8.0)	59.84 ^L+***(8.2)

SD: Standard deviation

EMS: Ethylmethane sulfonate

# Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-sided probabilities):

p values for comparisons with control using Williams’ test, unless indicated otherwise (+ t-test)

*** p < 0.001 (statistically significant)

L: Analysis performed upon logarithmically transformed data

Historical control data

Table 5: Mean tail intensity historical control data generated in the testing facility in Mar 2018 - Mar 2020 (liver and duodenum tissue) and in Dec 2011 - Mar 2020 (glandular stomach tissue), combined data generated in Sprague-Dawley and Wistar Han rats

Mean % Tail Intensity
Historical control data: liver tissue					Historical control data: duodenum tissue				Historical control data: glandular stomach tissue
	Vehicle Control Values		Positive Control Values		Vehicle Control Values		Positive Control Values		Vehicle Control Values		Positive Control Values
	172 Animals	30 Studies	87 Animals	29 Studies	124 Animals	22 Studies	66 Animals	22 Studies	71 Animals	12 Studies	54 Animals	12 Studies
	Individual	Group	Individual	Group	Individual	Group	Individual	Group	Individual	Group	Individual	Group
Minimum	0.9	1.5	15.9	18.4	1	1.3	27	29.4	1.5	2.4	28	34.4
Maximum	5.2	3.7	51.4	41.3	7.7	6.7	63	52.7	14.3	12.6	70.3	64.8
Mean	2.3	2.3	29.8	29.8	4	4	39.3	39.3	4.7	4.8	54.5	53.4
Standard Deviation	0.8	0.5	7.2	6.3	1.5	1.3	8.1	7.1	2.5	2.6	9.5	9.2
Lower 95% CL	0.7	1.3	15.3	17.1	1	1.3	23.1	25.1	0	0	35.5	35.1
Upper 95% CL	4	3.3	44.2	42.4	7.1	6.7	55.5	53.5	9.7	10.1	73.5	71.8

CL: Confidence limits

Table 6: Median tail intensity historical control data generated in the testing facility in Mar 2018 - Mar 2020 (liver and duodenum tissue) and in Oct 2017 - Mar 2020 (glandular stomach tissue), combined data generated in Sprague-Dawley and Wistar Han rats

Median % Tail Intensity
Historical control data: liver tissue					Historical control data: duodenum tissue				Historical control data: glandular stomach tissue
	Vehicle Control Values		Positive Control Values		Vehicle Control Values		Positive Control Values		Vehicle Control Values		Positive Control Values
	172 Animals	30 Studies	87 Animals	29 Studies	124 Animals	22 Studies	66 Animals	22 Studies	71 Animals	12 Studies	54 Animals	12 Studies
	Individual	Group	Individual	Group	Individual	Group	Individual	Group	Individual	Group	Individual	Group
Minimum	0	0.1	11.9	14.9	0.1	0.3	23.2	26	0.2	0.5	25	32.1
Maximum	1.4	0.7	52.6	40.9	4.1	3.2	65.4	53	3.9	2.6	63.4	56.8
Mean	0.4	0.4	27.4	27.4	1.4	1.3	37.5	37.5	1.2	1.2	43.6	43.6
Standard Deviation	0.2	0.2	8.4	7.4	1.1	0.9	9.5	8.2	1.2	1.2	14.4	12.5
Lower 95% CL	0	0.1	10.5	12.7	0	0	18.4	21	0	0	14.8	18.7
Upper 95% CL	0.8	0.7	44.3	42.1	3.5	3.1	56.5	54	3.6	3.6	72.5	68.5

CL: Confidence limits

Applicant's summary and conclusion

Conclusions:

Oral administration of the test substance within the present comet assay resulted in an increase in DNA strand breaks in the duodenum and glandular stomach of male Crl:Wistar Han rats. However, due to the confounding presence of hedgehog cells, these increases are considered to be linked with cytotoxicity. There were no treatment-related findings at histopathological examination. Based on the experimental findings, the test item is considered to be equivocal for DNA damage in the duodenum and glandular stomach, whereas it is negative for DNA damage in the liver.