Sodium O,O-di-sec-butyl dithiophosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

Combined repeated toxicity study with reproduction/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 June 2019 to 15 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650 Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium O,O-diethyl dithiophosphate

EC Number:

222-079-2

EC Name:

Sodium O,O-diethyl dithiophosphate

Cas Number:

3338-24-7

Molecular formula:

C4H11O2PS2Na

IUPAC Name:

Sodium O,O-diethyl dithiophosphate

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Han:Wistar of Wistar origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90 Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: 83-92 days; Females: 75-83 days
- Weight at study initiation: Males: 348-421 g; Females: 210-259 g
- Housing: Type III polypropylene/polycarbonate
Before mating: 2 animals of the same sex /cage.
Mating: 1 male and 1 female /cage.
Mated females: individually.
Males after mating: 2 animals /cage.
- Diet: Commercial ssniff® SM R/M-Z+H complete diet for rats and mice - breeding and maintenance and ssniff® SSNIFF rat/Souris Elevage E, 10 mm autoclavable complete feed for rats, ad libitum
- Water: Ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30-70 %
- Air changes (per hr): Above 10 air-exchanges/hour by a central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light

IN-LIFE DATES: From 26 June 2019 to 21 August 2019

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
VEHICLE:
- Concentration in vehicle: The test substance was formulated in the vehicle in concentrations of 30, 90 and 200 mg/mL.
- Amount of vehicle (if gavage): The dose volume was 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed three times during the study. Five aliquots of 5 mL of each formulation and five aliquots of control substance (vehicle) were taken and analyzed. The samples were stored at 2 - 8°C until analysis. Concentration of the test item in the dosing formulations varied between the range of 97.3 % and 105 % in comparison to the nominal values.

Duration of treatment / exposure:

Dosing of both sexes began after acclimatization and was continued up to and including the day before the necropsy.
Male animals were dosed for 44 days, females were dosed for 51 or 56 days.

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

450 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: See supporting study: 14-Day Oral Gavage Dose Range Finding Study with EP1-Na (Danafloat 123) in Rats, Toxi-Coop Zrt., 2019

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
- Time schedule: Twice daily.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: General clinical observations were made on parental animals once a day, after the administration.
Detailed examinations were made weekly until necropsy. Detailed clinical observations were made on all animals outside the home cage in a standard arena once prior to the first exposure and once weekly thereafter. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on five male and five female animals randomly selected from each group during the last exposure week. General physical condition and behavior of animals were tested.

BODY WEIGHT:
- Time schedule for examinations:
Parental males were weighed on the first day of dosing (Day 0) and weekly thereafter and on the day of the necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition), 4 and 13 post-partum. Body weight of the female animals was additionally weighed on gestational day 10 in order to give accurate treatment volumes, but these data were not evaluated statistically. Body weight was measured on the day of necropsy for female animals subjected to organ weighing (selected for further examinations).

FOOD CONSUMPTION:
The food consumption was determined weekly during the treatment period except mating phase (pre-mating days 0, 7, 13 and post-mating days 20, 27, 34 and 41 for male animals, pre-mating days 0, 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13 for female animals).

CLINICAL PATHOLOGY:
Clinical pathology examinations including hematology and clinical chemistry were conducted in five male and five female animals randomly selected from each group one day after the last treatment (i.e. on the day of necropsy). Animals were food deprived for approximately 16 hours (overnight) prior to blood collection.

HEMATOLOGY:
Parameters checked in Table 1 were examined.

BLOOD COAGULATION:
Parameters examined: Activated partial Thromboplastin Time (APTT), Prothrombin Time (PT)

CLINICAL CHEMISTRY:
Parameters checked in Table 2 were examined.

DETERMINATION OF SERUM THYROID HORMONE:
Blood samples for determination of serum levels of thyroid hormones (FT4 and TSH) were collected from animals as follows:
- from 2-8 pups per litter on post-natal day 4 (if it was feasible; samples were pooled by litter)
- from all dams and from 6-7 pups per litter on post-partum/post-natal day 13;
- from all parent male animals at termination on Day 44.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
At the time of termination, body weight, brain weight, weight of the testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole of all male adult animals were determined. In addition, for five males and females randomly selected from each group, adrenal glands, brain, heart, kidneys, liver, spleen and thymus were weighed.
The ovaries, uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, and all organs showing macroscopic lesions of all adult animals were preserved. Thyroid gland was preserved from all adult males and females and from one male and one female pup per litter. Tissues and organs (see Table 3) were collected from 5 male and five female animals randomly selected from each group.

Detailed histological examination was performed on the ovaries, uterus with cervix, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups – including non-pregnant female and its mating partner – with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose (1000 mg/kg bw/day) groups and in dead female animals at 450 mg/kg bw/day.
Additionally, organs and tissues were processed and evaluated histologically on the basis of necropsy observations.

Statistics:

The statistical evaluation was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible.
Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no test item related mortality at any dose level.
One dam at 450 mg/kg bw/day (1/12), was found dead on post-partum (lactation) day 5. There were no preceding clinical signs for this animal. Based on findings at necropsy and histological evaluation, the cause of death was judged to be individual disease and not related to the treatment as no death occurred at the higher (1000 mg/kg bw/day) dose level.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slightly reduced mean body weight gain in male and female animals at 1000 mg/kg bw/day resulted in minor changes in the mean body weight (≥ - 7 % relative to control) therefore was considered to have little or no toxicological relevance.

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross necropsy revealed slight damage of mucous membrane of the stomach in some female animals at 450 and 1000 mg/kg bw/day due to the local effect of the test item (thickened mucous membrane, erosion).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Squamous cell hyperplasia in the non-grandular part of the stomach was observed in some female animals at 450 and 1000 mg/kg bw/day (4/4 and 6/6 respectively)

Effect levels

1

Dose descriptor:

other: NOAEC

Effect level:

30 other: mg/mL (administered to animals of 150 mg/kg bw/day group)

Based on:

act. ingr.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Systemic toxicity

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

There were no signs of systemic toxicity in male or female animals at 150, 450 or 1000 mg/kg bw/day. In the female animals at 450 and 1000 mg/kg bw/day, squamous cell hyperplasia in the non-glandular part of the stomach referred to the local effect of the test item. Besides of this local tissue irritation no other histological findings were noted.

Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for systemic toxicity of male/ female rats: 1000 mg/kg bw/day
NOAEC (stomach irritation, female rats): 30 mg/mL (administered to animals of 150 mg/kg bw/day group)

Remark: The test substance as produced and marketet contains NaOH due to the production method.

Executive summary:

The toxicity of the substance was assessed in a Combined Dose Toxicity Study with the Reproductive/Developmental Screening Test according to OECD 422 (adopted 29 July 2016). Doses of 0 (control), 150, 450 and 1000 mg/kg bw/day was administered by gavage to 12 Wistar rats/sex/dose. Male rats were dosed daily for 44 days, female rats were dosed daily for 51 or 56 days.

 

No test item related mortality or clinical signs of systemic toxicity were observed in male or female animals during the study. Slightly reduced mean body weight gain in male and female animals at 1000 mg/kg bw/day resulted in minor changes in the mean body weight (≥ - 7% relative to control) therefore was considered to have little or no toxicological relevance. No changes in haematological or clinical biochemistry parameters were observed in either sex. The gross pathology showed no changes in organ weights compared to control group. Slight damage of mucous membrane of the stomach in some female animals at 450 and 1000 mg/kg bw/day were observed due to the local effect of the test item (thickened mucous membrane, erosion). In female animals at 450 and 1000 mg/kg bw/day, squamous cell hyperplasia in the non-glandular part of the stomach referred to the local effect of the test item. Besides of this local tissue irritation no other histological findings were noted.

 

Based on these observations the NOAEL for systemic toxicity of male/female rats was 1000 mg/kg bw/day.