Sodium O,O-diisobutyl dithiophosphate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium O,O-diisobutyl dithiophosphate

EC Number:

258-508-5

EC Name:

Sodium O,O-diisobutyl dithiophosphate

Cas Number:

53378-51-1

Molecular formula:

C8H18NaO2PS2

IUPAC Name:

sodium O,O-diisobutyl dithiophosphate

Test material form:

other: 51 % w/w solution in water, stabilized with X % NaOH

Details on test material:

- Substance type: Liquid
- Physical state: Light brown liquid
- Molecular formula (if other than submission substance): C8H18NaO2PS2
- Molecular weight (if other than submission substance): 264
- Smiles notation (if other than submission substance): CC(C)COP(=S)([S-])OCC(C)C.[Na+]
- InChl (if other than submission substance): InChI=1/C8H19O2PS2.Na/c1-7(2)5-9-11(12,13)10-6-8(3)4;/h7-8H,5-6H2,1-4H3,(H,12,13);/q;+1/p-1

Test animals

Species:

rat

Strain:

other: Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Males/females: Both 59 days
- Weight at study initiation: Males: 272.2 g to 299.9 g, Females: 210.4 g to 233.6 g

- Fasting period before study: 16 hours
- Housing: kept individually in MAKROLON cages (type III plus)
- Diet (e.g. ad libitum): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany; served as food ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg b.w./day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle mixtures samples of approx. 2 x 1 mL were taken at the following time points and stored at ≤ minus 20°C until analysis at LPT:

- Start of treatment period:
Concentration and stability:
Immediately after preparation of the test item vehicle mixtures as well as 8 and 24 hours after storage of the test item preparations at room temperature:
3 samples/dose level group (groups 2 to 4)
Number of samples: 3 x 3 = 9
Homogeneity:
At start of administration, during (middle) administration and before administration to the last animal of each dose level group:
3 samples/dose level group (groups 2 to 4).
Number of samples: 3 x 3 = 9

- End of treatment period:
Concentration:
During treatment with the test item always before administration to the last animal/dose level group:
1 sample/dose level group (groups 2 to 4).
Number of samples: TD 26 (1 x 3) = 3
Number of samples: TD 39 (1 x 3) = 3

Sum of all samples: 24, sum of all aliquots: 48

The samples were labelled with the study number, test item, test species, type of sample, aliquot number, concentration, test day, sampling time and date.

Duration of treatment / exposure:

Males: The daily administration of the test item started two weeks before mating and lasted until the day before sacrifice (considering a minimum total dosing period of at least 28 days).
Females: The daily administration of the test item started two weeks before mating and lasted up to at least day 3 of lactation.

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.:

60 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 male and 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: See supporting study: 14-DAY DOSE-RANGE-FINDING STUDY OF IBP1-Na (DANAFLOATTM 245) BY ORAL ADMINISTRATION TO RATS

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least daily, the frequency was increased when signs of toxicity were observed.
Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behavior patterns. The onset, intensity and duration of any signs observed were recorded.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioral changes, reaction to treatment or illness.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least daily, the frequency was increased when signs of toxicity were observed.
Additionally, once before the first exposure (to allow within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal was recorded on a weekly or daily basis throughout the experimental period with the exception of the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals from each group

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the premating period
- Animals fasted: Yes
- How many animals: 5 male and 5 female animals from each group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

HISTOPATHOLOGY: Yes

Statistics:

Toxicology and pathology data were captured, whenever possible, using the departmental computerized systems (Provantis® Integrated preclinical software, version 8.2.0.8, Instem LSS Ltd.). Raw data not fully compatible with the computerized
systems were maintained on paper according to appropriate SOPs. The test item-treated groups (2 to 4) were compared with the control group (1). The following statistical methods were used: STUDENT's t-test All numerical functional tests (p ≤ 0.01)
Multiple t-test based on DUNNETT, C. W. New tables for multiple Comparisons with a control Biometrics, 482-491 (Sept 1964) Body weight / Food consumption / Haematology / Clinical chemistry / Absolute and realtive organ weights (p ≤ 0.05 and p ≤ 0.01)
For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance was p ≤ 0.01. Exact test of R. A. FISHER Histopathology (p ≤ 0.05) For the comparison of classification measurements (for example the Fertility Index) the FISHER's exact test, n < 100 or chi2-test with Yates' correction for continuity, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01) were employed. These statistical procedures were used for all data. Significantly different data were indicated in the tables of the report. The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to  1 may occur caused by rounding.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At the intermediate and the high dose level (200 and 600 mg IBP1-Na/kg b.w./day) a slight to extreme salivation was noted in nearly all animals, with a higher incidence at the high dose level. Piloerection and an increased water consumption were noted for a few animals of the intermediate and the high dose group, which is most like related to the alkalinity of the substance.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related premature death was noted.
One male animal from the intermediate dose group (200 mg IBP1-Na/kg b.w./day) died prematurely on test day 32. The examination of the aninmal at autopsy and at histopathology revealed changes in the cardia part of the stomach (forestomach), which were also found in further animals of the intermediate and the high dose group and which are considered as to be test item related. However, the death of this animal was most likely referred to heart failure indicated by a severely increased heart weight. The death is therefore regarded to be spontaneous.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A slight reduction in body weight was noted until the end of the study on test day 38 for the male rats of the high dose group (600 mg IBP1-Na/kg b.w./day). Body weight at autopsy was reduced accordingly.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Examination of the haematological and biochemical parameters revealed statistically significant changes in the male and female animals of the high dose group (600 mg IBP1-Na/kg b.w./day), which were considered as to be test item-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males: A statistically significant increase in the plasma activity of ALAT and ASAT, a decrease of the chloride concentration and a decrease in the globulin concentration leading to an increase in the albumin/globulin ratio were noted in the high dose group.
Females: At the intermediate and the high dose level statistically significant dose dependent minor decreases of the chloride and sodium concentration were noted. At the high dose level the ASAT activity was increased.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

The neurological screening revealed a slight reduction for the hindlimb grip strength of the male and female animals of the high dose group (600 mg IBP1-Na/kg b.w./day).

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Examination of the organ weights revealed a slightly statistically significant increase in the relative liver weight of the male rats of the high dose group (600 mg IBP1-Na/kg b.w./day).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic inspection at autopsy revealed test item related changes in the cardia part of the stomach from animals of the intermediate dose group (200 mg IBP1- Na/kg b.w./day) and in the cardia part of the stomach from 1 female animal of the high dose group (600 mg IBP1-Na/kg b.w./day).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The histopathological examination revealed test item related changes in the forestomach from male and female rats of the intermediate and the high dose group (200 and 600 mg IBP1-Na/kg b.w./day) in form of hyperplasia and hyperkeratosis of the squamous cell epithelium, infiltration with neutrophilic granulocytes, granulation tissue and ulcerations.
Additionally, test item-related changes were noted in the liver in the form of a slight centrilobullar hepatocellular hypertrophy at 600 mg/kg bw/day.

Effect levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: systemic effects

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The following no-observed-adverse-effect levels were noted for the test substance:

Effects on the F0-generation
NOAEL (no-observed-adverse-effect level): 200 mg IBP1-Na/kg b.w./day, p.o.

Effects on reproduction
NOAEL (no-observed-adverse-effect level): 600 mg IBP1-Na/kg b.w./day, p.o.

Remark: The test substance as produced and marketed 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 22 march 1996). Doses of 0 (control), 60, 200 and 600 mg/kg bw/day was administered by gavage to 10 Crl: CD(SD rats/sex/dose. Male rats were dosed daily for 37 days, female rats were dosed daily starting two weeks before mating and continued to a least day 3 of lactation.

The following no-observed-adverse-effect levels were noted for the test substance:

Effects on the F0-generation
NOAEL (no-observed-adverse-effect level): 200 mg IBP1-Na/kg b.w./day, p.o.

Effects on reproduction
NOAEL (no-observed-adverse-effect level): 600 mg IBP1-Na/kg b.w./day, p.o.