Sodium O,O-diisobutyl dithiophosphate

Endpoint summary

Description of key information

OECD 422:

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.

OECD 408:

The no-observed-adverse effect level (NOAEL) was established at 600 mg/kg bw/day in males and female rats.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

60 mg/kg bw/day (nominal)

Dose / conc.:

200 mg/kg bw/day (nominal)

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

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.

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.

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

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:approx. 6-7 weeks old
- Weight at study initiation:
males: 177 – 205 g (mean: 190.43 g, ± 20% = 152.34 – 228.51 g)
females: 132 – 155 g (mean: 146.53 g, ± 20% = 117.22 – 175.83 g)

- Fasting period before study:
- Housing: - Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): - Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum):- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
- Diet (e.g. ad libitum): - Free access to Altromin 1324 maintenance diet for rats and mice
- Water (e.g. ad libitum):- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control
ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: February 25th 2016 (study initiantion date) To: June 9th 2016 (experimental completion date)
Experimental Starting Date: March 2nd 2016
Treatment Period:March 8th 2016 to June 8th 2016

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 90 days. Animals of an additional control group were handled identically as the dose groups but received aqua ad iniectabilia (sterile water), the vehicle used in this study. The 4 groups comprised of
10 male and 10 female Wistar rats.
The following doses were evaluated:
Control: 0 mg/kg bw/day
Low Dose: 30 mg/kg bw/day
Medium Dose: 150 mg/kg bw/day
High Dose: 600 mg/kg bw/day
The test item formulations were used not more than 10 days after preparation. The test item was dissolved in aqua ad iniectabilia and administered daily during a 90-day treatment period to male and female animals by oral gavage. For each animal, the individual dosing volume was calculated on the basis of the body weight most recently measured and administered at approximately the same time each day.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

The test item formulations were used not more than 10 days after preparation

The test item was dissolved in aqua ad iniectabilia and administered daily during a 90-day treatment period to male and female animals by oral gavage.

For the preparation of test item formulations, the required amount of test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item. The mixture was stirred continuously for ca 1 minute, the amount of test item required to achieve the targeted dose concentration of active ingredient within the formulation sample was calculated based on the concentration of active ingredient within the test item (51.0%).

VEHICLE
- Concentration in vehicle:
Control: 0 mg/kg bw/day
Low Dose: 30 mg/kg bw/day
Medium Dose: 150 mg/kg bw/day
High Dose: 600 mg/kg bw/day

- Amount of vehicle (if gavage): For each animal, the individual dosing volume was calculated on the basis of the body weight most recently measured and administered at approximately the same time each day.The application volume for all groups was 5 mL/kg body weight
- Lot/batch no. (if required):
Name: aqua ad iniectabilia
Manufacturer: AlleMan Pharma
Batch No.: 503424
Physical State: liquid
Storage Conditions: room temperature
Expiry Date: 02/2018

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In the 1st, 5th and 13th week of treatment, samples for the testing of homogeneity were taken from the top, middle and bottom of the freshly prepared high and low dose formulations (in total 18 samples).
For determination of the concentration of test item in dosing formulations, samples from all dose groups were retained in week 1, 5, 9 and 13 of the treatment period. Hence, in total 16 samples were taken for determination of concentration.
Each sample was retained in duplicate (sample A and B) with a volume of approx. 5 mL. All samples were stored between -15 °C and -35 °C. The ‘A’ samples were delivered to Eurofins Munich (Test Site 3) for analysis. The samples ‘B’ were retained at BSL Munich as back-up until the analyses of samples ‘A’ have been performed.
Concentration results were considered acceptable as sample concentration results were within or equal to ± 10% of theoretical concentration. For homogeneity, the criteria for acceptability were a relative standard deviation (RSD) of concentrations of ≤10% for each group.
All samples of dosing formulations were analysed at Test Site 3 in accordance with GLP under the study reference number 156098. All samples were discarded after completion of the final study report.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group, referred to as Control

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

Low dose, referred to as LD

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Medium dose, referred to as MD

Dose / conc.:

600 mg/kg bw/day (nominal)

Remarks:

High dose, referred to as HD

No. of animals per sex per dose:

10 male and 10 female animals per group

Control animals:

yes

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes


DETAILED CLINICAL OBSERVATIONS: Yes

Clinical Observations
All animals were observed for clinical signs during the entire treatment period of 90 days.
General clinical observations were made at least once a day, preferably at the same time each day and considering the peak period of anticipated effects after dosing (based on information from previous studies. The health condition of the each animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed observations with focus on considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.
Ophthalmological examinations, using an ophthalmoscope were made on all animals before the first administration and during the last week of the treatment period.

Functional Observations
Once before the first exposure and once in the last week of exposure multiple detailed behavioural observations were made outside the home cage using a functional observational battery of tests [11]. These tests were conducted in all animals .
The procedure applied to each animal was briefly as follows: After observing the animal in the home cage, it was transferred to the open field and watched for 2 minutes. Thereafter animal was removed from the arena and manipulated to assess reflexes, grip strength, pupil response and reactions to handling were tested and approx. 10 minutes after returning the animal to the home cage, body temperature was measured rectally. During the length performance of this procedure data of on a battery of 47 motor-behavioural, visceral and sensory parameters were assessed and collected.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Not specified
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematological parameters were examined in blood collected at the end of the treatment prior to or as part of the sacrifice of the animals
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 3 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animal
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 5 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:A urinalysis was performed with samples collected from all animals prior to or as part of the sacrifice of the animals
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters checked in table 6 were examined.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 8)
Gross necropsy
One day after the last administration (study day 91) all surviving animals of the treatment period following blood sampling were sacrificed using anesthesia (by injection of ketamine and xylazin solution into the inferior vena cava) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities
Organ Weight
The wet weight of the selected organs (seen in Table 8) of from all sacrificed animals was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead or euthanised for animal welfare reasons were not taken.

HISTOPATHOLOGY: Yes (see table 9)
The tissues (in Table 9) from all animals were preserved in 4% neutral-buffered formaldehyde except eyes, testes and epididymides which were fixed in Modified Davidson’s fixative for approximately 24 hours before they were transferred to 70% ethanol.

Statistics:

Evaluation of Results and Statistical Analysis
The findings of this study were evaluated in terms of the observed effects.
Parameters like body weight gain and food consumption were calculated for each
animal as the difference in weight measured from one week to the next. Mean group
body weights were calculated.
The individual and mean group relative organ weights were calculated in relation to the
brain weight and in relation to the body weight (measured at necropsy) and are
presented as percentage.
All results are reported in a tabular form (as group mean in summary tables and/or listed
in individual data tables) (Annex 1 in the full study report).
Analytical results and histopathological findings are presented in separate phase-reports
attached to this report (Annex 2 and Annex 3 in the full study report).
With few exceptions, toxicology and pathology data were captured, using the validated
departmental computerized system Ascentos® System (version 1.1., Pathology Data
Systems Ltd.). Otherwise, raw data were recorded on paper according to appropriate
SOPs.
A statistical assessment of the data on body weights, food consumption, parameters of
haematology, blood coagulation and clinical biochemistry and absolute and relative
organ weights were performed for each gender by comparing values from dosed
animals with control animals using either a parametric one-way ANOVA and a post-hoc
Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test,
based on the results of homogeneity and normality tests. Statistical analyses were
performed with Ascentos 1.1.3 software or GraphPad Prism V.6.01 software (p<0.05 is
considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related adverse observations in either sex.
The following clinical findings were recorded during the study:
Salivation and moving the bedding were observed in MD and HD animals, immediately after administration with a roughly similar incidence among those groups. These clinical findings, closely related to the administration procedure, indicate a local effect of the test item formulation and are not considered as signs of adverse systemic effects. Similar findings were not observed in males and females of the LD and C groups.
In addition, on one single day in one female and on two occasions in another female, both from the HD group, abnormal breathing was recorded after dosing. Alopecia was also observed in 2 females of LD group for 6 days. As these effects were observed only on few days and/or with no dose response, they are considered not to be toxicologically relevant.
With the exception of moribund condition of animal no 32 (male HD,) no other clinical observations were recorded during the daily observations.
During the course of the study, the following parameters of the weekly detailed clinical observations were statistically different to the control in males: salivation higher in HD in week 6 (HD 4.7, control 4.0), in week 7 (HD 4.9, control 4.0), in week 8 (HD 6.0, control 4.0), in week 9 (HD 4.9, control 3.7), in week 10 (HD 4.9, control 4.0) and in week 11 (HD 5.1, control 4.0), spontaneous activity lower in HD in week 7 (HD 3.6, control 4.0), in week 8 (MD 3.2, HD 2.6, control 3.9) and in week 9 (HD 3.6, control 4.0). Furthermore, in week 3, lower animal sleeps score in MD and HD (MD 0.0, HD 0.0, control 0.5) and higher animal moving in the cage score in MD and HD (MD 1.0, HD 1.0, control 0.5) was observed.
The following parameters of the weekly detailed clinical observations were statistically different to the control in females: salivation higher in HD in week 4 and 5 (HD 4.6, control 4.0), in week 7 (HD 5.0, control 4.0), in week 8 (HD 5.2, control 4.0), in week 9 (HD 6.0, control 4.0) and in week 10 (HD 5.4, control 4.0), spontaneous activity lower in MD and HD in week 9 (MD 3.4, HD 3.2, control 4.0) and in week 10 (HD 3.5, control 4.0). Furthermore, in week 8, lower animal sleeps score in LD, MD and HD (LD 0.0, MD 0.0, HD 0.0, control 0.3) and higher animal moving in the cage score in LD, MD and HD (LD 1.0, MD 1.0, HD 1.0, control 0.7) was observed.
All other detailed clinical observation parameters were within the normal range of variation of historical control data for this strain. Any statistically significant differences between dose and control groups are not assumed to be biologically relevant.
There were no ophthalmoscopic findings in any of the animals of this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There was no treatment-related mortality in either sex.
Animals no. 31 (male HD) was found dead on day 84 and male no. 32 (male HD) was euthanized for ethical reasons on day 35.
Animal no. 70 (female MD) was found dead on day 12 but was almost completely cannibalised by the cage mates and therefore no tissues were available for histopathology.
Animal no. 32 was euthanized in a moribund condition. Until this condition occurred clinical signs like abnormal breathing, salivation, piloerection and hunched posture had been observed for a week. Although the gross lesions recorded in animals nos. 31 and 32 (affections of the thoracic cavity) were indicating a gavage error, the histological evaluation of the organs and tissues did not reveal the cause of morbidity/death of these animals.
The cause of death of the animal no. 70 could not be established as no tissues were available for histopathology.
Apart from that no mortality occurred in the control or any of the dose groups during the treatment and recovery period of this study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In both males and females, the mean body weight increased with the progress of the study in all groups.
In males, the mean body weight was 10-11 % higher in the HD group from day 36-90 without achieving statistical significance. On day 90 the difference in body weight of the HD males to controls reached 11.02 %. This body weight increase is considered to be a test item related. Moreover increase in MD male body weight was also observed but this increase was marginal (0-4.68 %).
In females, the mean body weight was 5-8 % higher in the HD group from day 36-90 and statistical significance was achieved on day 50 and 64 when compared with the controls. This mean body weight was within the expected historical range of variation with no considerable differences between dose groups and control group during the treatment period.
In HD males the overall body weight change from day 1 to 90 was higher without achieving statistical significance compared to the controls. However, statistically significantly higher body weight change was observed during day 85-90 in MD and HD groups when compared with the controls.
In accordance with body weight development, in females, the weight gain was within the expected historical range of variation. However, overall body weight change from day 1 to 90 was marginally but statistically significantly higher in MD and HD group when compared with the controls.
Therefore these finding in HD males but not females represent test item relation. However, toxicological significance of this increase in body weight gain in males was not clear.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In correlation to body weight gain, the food consumption in males and females was higher at the beginning of the study and then constantly decreased with the progress of the study in all groups, which is considered to be a normal development as food demand is usually highest at a young age and then constantly decreases. During the whole treatment period, food consumption of the male and female HD group was higher compared to the corresponding control group, which correlates to the higher mean body weight and body weight gain of the male and females in these groups.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In female, statistically significantly higher WBC (MD 3.436, HD 3.750 and control 2.065) and PLT (MD 851.8, HD 855.6 and control 697.5) counts were observed in MD and HD group when compared with the controls. These statistically significant differences between dose and control groups in females are not assumed to be biologically relevant as no such effect was observed on differential WBC count and values for both WBC and PLT were within the normal range of variation of historical control data.
All other haematology parameters in males and females were within the normal range of variation of historical control data for this strain.
Blood coagulation was not affected by IBP1-Na. The observed values were within the normal range of variation of historical control data.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In the female, statistically significantly lower ALAT (17.97 % of control) and ASAT (20.31 of control) were observed in HD group when compared with the controls. These statistically significant differences between dose and control groups in females are not assumed to be biologically relevant as decrease in these liver enzymes have no biological significance and values for both ALAT and ASAT were within the normal range of variation of historical control data.
Besides, all parameters of clinical chemistry in males and females were within the normal range of variation of historical control data for this strain and are not assumed to be toxicologically relevant.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Slightly high protein and erythrocytes levels were found in the urine of few male and females of all groups including controls. As this effect was observed in animals of all groups including control, it was considered to be isolated findings with no toxicological relevance.
All other parameters of urinalysis in test item treated males and females at the end of the treatment period were not considerably different to the corresponding control and were within the normal range of variation of historical data for this strain.

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period in HD group of both, males and females, a moderate statistically significantly higher mean absolute and relative (to body weight) weight of liver was recorded, compared to the corresponding control (mean absolute/relative +29%/+18% in males and +13%/+9% in females). In histology, this effect was associated with centrilobular hepatocellular hypothrophy in both genders. There were no further indicators for toxicity in any of the organs and tissues examined. Therefore, this finding is not considered to be of adverse nature, but related to increase in liver metabolism.
The kidney of HD males but not females was statistically significantly higher with mean absolute organ weight value of 21% above the C group. Histopathologically, there were minor changes characterized by minimal tubular dilation associated with a swelling (vacuolation) of the epitehlia and such lesions are in general reversible. There were no findings indicating a morphological injury (see also histopathology phase report) and therefore this effect on male kidney weight was not considered to be adverse.
In HD males, a statistically significantly higher absolute mean weight of prostate with seminal vesicles and coagulation glands (3.38 g in HD vs 2.80 g in C) was recorded. In the light of the absence of histopathological effect, this effect on prostate with seminal vesicles and coagulation glands weight was not considered to be adverse.
Besides, all male and female organ weights were within the normal range of variation of historical control data for this strain and differences between dose and control groups are not assumed to be biologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pathological changes were observed in the following animals. Abnormal red color and fluid filled thoracic cavity was observed in male no. 31 (HD). The thymus showed abnormal dark color in male no. 32 (HD). There was also black focus on lung and gas filled stomach and gastrointestinal tract was observed in male no. 32. Histopathologically, lung finding was due to congestion and for stomach finding, no histopathological correlate was observed.
The spleen of female no. 66 (MD) was reported to be enlarged. In histology, this finding correlates with congestion. There was also a cyst on ovary found in female no. 55 of LD group for which no histopathological correlate was observed.
Fluid filled uterus with cervix was recorded in 2/10 LD females (no. 51 and 54), 2/10 MD females (no. 63 and 65) and 2/10 HD females (no. 71 and 80). In histology, this finding correlates with female cyclic change and represents a normal background finding for this strain.
Female no. 70 could not be observed for macropscopic findings as the animal was completely cannibalised by the cage mates.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of this study, there were three decedents at 600 mg/kg. The cause of morbidity/death could not be established.
Gross lesions were not remarkable, however, histopathology revealed lesions in the liver and kidneys 8 males only) at a minor severity. Secondary lesions were noted in the adrenal glands and thymus.
In the liver, there was hepatocellular hypertrophy (mainly centrilobular) at minor severity degrees at 600 mg/kg in both sexes. There were no further indicators for toxicity in any of the organs and tissues examined. Therefore, this finding is not considered to be of adverse nature, but related to increase liver metabolism.
In male kidneys at 600 mg/kg, there were minor changes characterized by minimal tubular dilation associated with a swelling (vacuolation) of the epitehlia, mainly in the rectal and distal tubules. The nature of this lesion is unknown but is likely related to storage in tubular cells and subsequent water uptake/disturbed release. Such lesions are in general reversible. There were no findings indicating a morphological injury.
Secondary findings, deemed to be stress-related consisted of diffuse cortical hypertrophy (zona fasciculata) at a minor severity in both sexes at 600 mg/kg, and a minimally increased thymic atrophy in males only.

In general, these findings are minor in severity and not considered to be adverse. Therefore, the NOAEL may be established at 600 mg/kg/day.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

600 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

not specified

Conclusions:

On the basis of the present study, the 90-Day Repeated Dose Oral Toxicity study with test item in male and female Wistar rats, with dose levels of 30, 150, and 600 mg/kg body weight day the following conclusions can be made:
No effect of toxicological relevance was observed on male and female body weight, food consumption, haematology, clinical biochemistry, urine parameters, clinical signs, gross pathology and organ weight up to 600 mg/kg bw/day.
The histomorphological no-observed-adverse effect level (NOAEL) was established at 600 mg/kg bw/day in males and female rats.
Based on the study findings, the overall NOAEL of test item for systemic toxicity in this study is considered to be at 600 mg/kg bw/day.

Executive summary:

The substance was investigated in a 90-Day Repeated Dose Oral Toxicity Study according to OECD Guideline 408 (adopted 21 September 1998). Doses of 0 (control), 30, 150 and 600 mg/kg bw/day was administered by gavage daily over a period of 90 days to 10 Wistar rats/sex/dose.

No effect of toxicological relevance was observed on male and female body weight, food consumption, hematology, clinical biochemistry, urine parameters, clinical signs, gross pathology and organ weight up to 600 mg/kg bw/day. Indication for stomach irritation (Lymphoid follicles, Incr.inflamm.infilt., vacuolation and inflammation) was noted in the high dose females and to a less extend at high dose males.
The histomorphological no-observed-adverse effect level (NOAEL) was established at 600 mg/kg bw/day in males and female rats.

Based on the study findings, the overall NOAEL of IBP1-Na for systemic toxicity in this study is considered to be at 600 mg/kg bw/day.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

high quality

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:


Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; digestive: stomach

Justification for classification or non-classification

NOAEL is well above classification criterion according to Regulation (EC) No 1272/2008.