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Toxicological information

Repeated dose toxicity: oral

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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:
2 (reliable with restrictions)
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

Constituent 1
Chemical structure
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 phosphorodithioate
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):
- 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 28 (1 x 3) = 3
Number of samples: TD 43 (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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bodyweight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
60 mg/kg bodyweight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bodyweight/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
600 mg/kg bodyweight/day
Basis:
actual ingested
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.
Cageside observations included skin/fur, eyes, mucous membranes, respiratory
and circulatory systems, somatomotor activity and behaviour 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 behavioural 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 behaviour (e.g. selfmutilation,
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 / No / No data
- Time schedule for examinations:

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes / No / No data
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (identity) / No / No data
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood:
- Animals fasted: Yes / No / No data
- How many animals:
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes / No / No data
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / No data
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)
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):
No test item-related premature death was noted
Mortality:
mortality observed, treatment-related
Description (incidence):
No test item-related premature death was noted
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 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 gr.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
The neurological screening revealed a 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).
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 centrilobullar hepatocellular hypertrophy at 600 mg/kg bw/day.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Systemtic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion
The following no-observed-adverse-effect levels were noted for the read-across 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): 200 mg IBP1-Na/kg b.w./day, p.o.

Remark: the test solution contains NaOH due to the production method.