Registration Dossier

Administrative data

Description of key information

A combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (BASF AG, 2003) adequately adresses the repeated exposure with IBDU. NOAELs were 300 mg/kg bw/day for females (reduced body weight gain during pregnancy and lactation at 1000 mg/kg bw/day), and 1000 mg/kg bw/day for males (highest tested dose level).

A subchronic repeated dose study according to OECD 408 (BASF SE, 2017) revealed a NOAEL for general systemic toxicity of 1000 mg/kg bw/day for male and female Wistar rats taking into account that tubular damage in the kidneys of male Wistar rats were a consequence of an α-nephropathy which does not represent a risk for humans.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 Sep 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicity and Ecology, 67056 Ludwigshafen, Germany.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: 2015-03-24
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
The rat is a frequently used laboratory animal, and there is comprehensive experience with this animal species. Moreover, the rat has been proposed as a suitable animal species by the OECD and the EPA for this type of study.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models GmbH, Sulzfeld, Germany.
- Age at study initiation: 42 ± 1 days
- Weight at study initiation (mean): Males:164.4 g; Females: 128.6 g
- Fasting period before study: No
- Housing: 5 animals per cage in polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm²)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Yes, 8 days.

DETAILS OF FOOD AND WATER QUALITY:
- Food analyses: The supplier assayed the food used in the study for chemical and microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable. Fed. Reg. Vol. 44, No. 91of 09 May 1979, p. 27354 (EPA), served as a guideline for maximum tolerable chemical contaminants. The number of microorganisms did not exceed 1 × 10^5/g food.
- Drinking water analyses: The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and by the Environmental Analytics Water/Steam Monitoring Department of BASF SE as well as for the presence of microorganisms by a contract laboratory. On the basis of the analytical findings, the drinking water was found to be suitable. German “Trinkwasserverordnung” (Drinking Water Regulation) served as a guideline for maximum tolerable contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod: 12 h light/ 12 h dark

Route of administration:
oral: gavage
Vehicle:
other: Drinking water containing 0.5% Carboxymethylcellulose
Details on oral exposure:
The test substance was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water containing 0.5% Carboxymethylcellulose was filled up to the desired volume and subsequently homogenized with an Ultraturrax. During administration, the test substance preparations were kept homogeneous by stirring with a magnetic stirrer. The test-substance preparations were produced once a week, at least. The administration volume was 10 mL/kg body weight.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
Homogeneity and concentration control analyses are performed in separate GLP studies but not all of them were completely finalized by 06 Feb 2017. Thus, an assessment is currently not possible.
The report of the present study was finalized without the results of these analyses because of urgent authority submission requirements of toxicology results. The lack of completeness of analytical results does not have an impact on the outcome of this toxicology study. After complete finalization of all analytical reports, they will be amended in a GLP-compliant Amendment to the Report for immediate submission to the Sponsor and to the authority.

The concentrations in the endpoint study record and the endpoint summary are thus currently the nominal concentrations.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Cage side observations checked in table [No.1] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the administration period and thereafter at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of the administration period on day -1 and on study day 84
- Dose groups that were examined: Control and high-dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the administration period
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- Parameters checked in table [No.4] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes
- Parameters checked in table [No.5] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the administration period
- Parameters checked in table [No.6] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / motor activity
- Parameters checked in table [No 2, 3] were examined
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 7)

HISTOPATHOLOGY: Yes (see table 8)
Other examinations:
In addition to the required examinations, special attention was given to the reproductive organs of male and female animals.

Estrous cycle determination:
Estrous cycle length and normality were evaluated daily for all female animals for a minimum of 3 weeks prior to necropsy.

Sperm parameters:
After organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and cohort 1A males; Sperm motility, Sperm morphology, Sperm head count (cauda epididymis) and Sperm head count (testis).
Statistics:
STATISTICS OF CLINICAL EXAMINATIONS
- Comparison of each group with the control group using DUNNETT's test (two-sided) for the hypothesis of equal means: Body weight, body weight change
- Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the equal medians: Rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity, estrous cycle, Bood parameters, Urine pH, volume and specific gravity, weight parameters
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians. In case of exactly the same numbers of the dose group and the control, no statistical test was performed: Urinalysis parameters,
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians; If only control and one dose group are measured, WILCOXON-test (onesided) without adjustment were used. For the percentage of abnormal sperms values < 6% were set to 6% (cut off 6%): Sperm analysis parameters
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Slight salivation shortly after treatment was observed in 7 male and 8 female animals of test group 3 (1000 mg/kg bw/d) on several days of the study. From the temporary, short appearance immediately after dosing it was concluded that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. No test substance-related effects were obtained in test groups 1 and 2 (100 and 300 mg/kg bw/d).
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance-related impairment in body weight parameters were observed for male and female animals in test groups 1-3 (100, 300 and 1000 mg/kg bw/d). Mean body weight of male animals in test group 3 (1000 mg/kg bw/d) was slightly but not significantly lower towards the end of the administration period, with a maximum of -4.2% on study day 91. The same was true for female animals of test group 2 (300 mg/kg bw/d), showing a maximum deviation of -6.5% on study day 77. These findings were assessed to be incidental and not related to treatment. Mean body weight change values were significantly decreased in female animals of test groups 2 (300 mg/kg bw/d) study day 77. The change was assessed to be incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption of male animals in test group 3 (1000 mg/kg bw/d) was slightly lower, with a maximum reduction of -6.0% at the end of the administration period. Due to the marginal occurrence, these changes were assessed as being incidental and not related to treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All apparent findings were assessed as being incidental in nature since they occurred in control as well as in treated animals and did not show a dose-response relationship.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
After the three months administration period in males of test group 2 (300 mg/kg bw/d) mean corpuscular volume (MCV) was lower compared to controls, but it was not dose dependently changed.
In males of test groups 1 and 3 (100 and 1000 mg/kg bw/d) absolute monocyte counts were higher compared to controls, but the values were within the historical control range (absolute monocytes 0.07-0.15 Giga/L).
Therefore, both alterations were regarded as incidental and not treatment-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes among clinical chemistry parameters were observed.
After the three months administration period in females of test groups 1 and 3 (100 and 1000 mg/kg bw/d) urea levels were increased. The mean of test group 3 was within the historical control range, that one of test group 1 above this range (urea 5.31-7.10 mmol/L). However, the urea level was not dose-dependently changed. In females of test group 3 (1000 mg/kg bw/d) sodium levels were lower compared to controls, but the values were within the historical control range (sodium 139.8-145.4 mmol/L). Therefore, both alterations were regarded as incidental and not treatment-related.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related, adverse changes among urinalysis parameters were observed.
In females of test group 3 (1000 mg/kg bw/d) urine volume was decreased and urine specific gravity was increased. These findings without any other changes of kidney parameters reflect the physiological adaptation of the kidneys towards lesser fluid income and, therefore, were regarded as adaptive and not adverse.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered to have been incidental. The following examinations were performed during FOB and were assessed individually:
- Home cage observations
- Open field observations
- Sensorimotor tests/reflexes
- Quantitative parameters
Overall, no test substance related effects were observed.

Motor activity measurement:
Regarding the overall motor activity as well as single intervals, no test substance-related deviations to the control animals were noted for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d). At interval No. 7 a decreased value was measured for male animals of test group 1 (100 mg/kg bw/d). The individual deviation was assessed not to be related to treatment as no dose-response relationship occurred.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute organ weight:
The absolute organ weights are listed in table 1 in section “any other information on results incl. tables”. The decreased absolute mean brain and epididymis weights in male animals of test group 3 (1000 mg/kg bw/d) were regarded as incidental as there were no changes in relative weights and no histopathological correlate. The decreased mean absolute and relative pituitary gland weights in female animals of test group 2 (300 mg/kg bw/d) were regarded as incidental as there was no dose-response and weights in test group 3 were identical to control values (100%).

Relative organ weights:
The relative organ weights are listed in table 2 in section “any other information on results incl. tables”. Relative weights of the liver in male animals of test group 3 (2.307%) were slightly above historical controls (2.110-2.299%). This was assumed to be caused by a change in mean body weight in this group rather than an effect on the liver, i.e. weight increase. Historical controls for terminal body weights of male animals range from 352.84 to 386.52 g. The terminal body weights of test group 3 animals in this study were slightly below this value (352.77 g). This was assumed to be the reason for the slightly increased relative liver weights in test group 3 animals (Absolute liver weights were within historical controls. Historical controls: 7.611-8.493 g, test group 3: 8.135 g). Furthermore, there was no dose-response and no histopathological correlate in test group 3. The increased relative liver weights in male animals of test group 3 were therefore regarded as incidental. All other mean relative weight parameters did not show significant differences when compared to the control group 0.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the kidneys of male animals there was a dose- and treatment-related increase in incidence and severity in eosinophilic droplets visualized by CAB staining, which was accompanied by an increased incidence of basophilic tubules (mostly of minimal severity) and, in test group 3, by tubular casts. Incidences and grading are shown in the table 3 in section "Any other information on results incl. tables".
In females, 3 animals of test group 3 (1000 mg/kg bw/d) showed a tubular cast. This was, however, unilateral and CAB staining for protein was negative in tubules. Therefore, these findings were regarded as incidental. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Estrous cycle:
No test substance-related effects on estrous cycle length and the number of cycles were obtained.

Sperm parameters:
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis, no treatment-related effects were observed.
Details on results:
The following test substance related, relevant findings were noted:

Test group 1 (100 mg/kg bw/d)
Clinical Examinations, Clinical Pathology and Pathology:
- No treatment-related, adverse effects were observed.

Test group 2 (300 mg/kg bw/d)
Clinical Examinations, Clinical Pathology and Pathology:
- No treatment-related, adverse effects were observed.

Test group 3 (1000 mg/kg bw/d)
Clinical Examinations and Clinical Pathology:
- No treatment-related, adverse effects were observed.
Pathology:
- Increased protein storage in tubules (eosinophilic droplets, positive CAB staining), increased incidence of basophilic tubules as compared to controls, and tubular casts in male animals (α2u-nephropathy)
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: General systemic toxicity
Remarks on result:
other: Tubular damage in the kidney of male Wistar rats was a consequence of an α2u-nephropathy which does not represent a risk for humans.
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: tubular damage in the kidneys (consequence of an α2u-nephropathy which does not represent a risk for humans)
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: General systemic toxicity
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Table 1: Absolute organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Brain

99%

96%

97%*

 

 

 

Epididymides

96%

95%

91%**

 

 

 

Pituitary gland

 

 

 

98%

86%**

100%

*p≤0.05; **p≤0.01

Table 2: Relative organ weights

 

Male animals

Female animals

Test group (mg/kg bw/d)

1

(100)

2

(300)

3

(1000)

1

(100)

2

(300)

3

(1000)

Liver

101%

101%

106%**

 

 

 

Pituitary gland

 

 

 

99%

90%*

100%

*p≤0.05; **p≤0.01

Table 3: Histopathology

 

Male animals

Test group (mg/kg bw/d)

0

(0)

1

(100)

2

(300)

3

(1000)

No. of animals

10

10

10

10

CAB staining

7

9

9

10

-         Grade 1

6

7

6

4

-         Grade 2

1

2

3

6

Tubules, basophilic, multifocal

4

6

6

9

-         Grade 1

3

6

6

7

-         Grade 2

1

 

 

2

Cast, tubular

1

1

 

9

-         Grade 1

1

1

 

6

-         Grade 2

 

 

 

2

-         Grade 3

 

 

 

1

Conclusions:
The oral administration of N,N'-(isobutylidene)diurea by gavage to male and female Wistar rats for 3 months caused adverse signs of toxicity in male animals, only, paying attention to
the tubular damage in the kidneys at 1000 mg/kg bw/d.
However, the no observed adverse effect level (NOAEL) for general systemic toxicity was set to 1000 mg/kg bw/d for male and female Wistar rats taking into account that tubular damage in the kidneys of male Wistar rats were a consequence of an alpha2u-nephropathy which does not represent a risk for humans (Durham and Swenberg, 2013).
Executive summary:

N,N'-(isobutylidene)diurea was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (test group 0), 100 (test group 1), 300 (test group 2) and

1000 mg/kg bw/d (test group 3) over a period of 3 months.

With regard to clinical examinations, no signs of general systemic toxicity were observed even at a dose level of 1000 mg/kg bw/d. In addition, no test substance-related effects on

estrous cycle length and the number of cycles were obtained.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

Regarding pathology, treatment-related findings were observed in the kidneys of male animals consisting of increased protein storage in tubules (eosinophilic droplets, positive CAB staining), an increasing incidence of basophilic tubules of minimal severity with dose, and in test group 3, tubular casts. In a previous study with this test item, the tubular protein was identified as alpha 2 urinary protein (BASF study No 99P0113/00132).

Findings were regarded as treatment-related and adverse in test group 3. Findings in test groups 1 and 2 were regarded as treatment-related but in absence of degenerative findings (tubular casts) as non-adverse.

Although the increase of eosinophilic droplets was considered treatment-related and adverse in test group 3 due to tubular damage, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013).

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: males: 8 weeks; females: 10 weeks
- Weight at study initiation: males: 276 g mean bw; females: 228 g mean bw
- Housing: housed individually in suspended wire-mesh cages, females shifted 4 days before lactation to polycarbonate cages in a barrier rodent unit.
- Diet: A04 C pelleted diet ad libitum, distributed weekly
- Water: tap water ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +- 2
- Humidity (%): 50 +- 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The vehicle was 0.5% aqueous carboxymethylcellulose solution prepared using:
- purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
- carboxymethylcellulose, batch No. 69H0028, supplied by Sigma (Saint-Quentin-Fallavier, France).

The test substance was administered as a suspension in the vehicle.
The test substance was ground using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 10, 30 and 100 mg/mL and then homogenized using a magnetic stirrer.
The test substance dosage forms were made daily (preparation stable for 3 hours).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses of the test substance preparations: concentration and homogeneity.
Three samples of each control and test substance dosage form (top, middle and bottom of the flasks ) prepared for use during the first week and the last week of treatment were taken. They were stored at -20°C pending dispatch, to the Sponsor, for analysis of concentration and homogeneity
Duration of treatment / exposure:
Male: throughout pre-mating (15 days), and during the mating and post-mating periods until sacrifice (34 days in total).
Female: throughout pre-mating (15 days) and mating period, during pregnancy and lactation, until day 4 post partum.
Frequency of treatment:
daily, 7 days/week
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 male and 10 female animals per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were specified by the Sponsor, following the results of a previously conducted prenatal developmental toxicity study in Wistar rats (BASF Project No. 30R0727/90116). The oral route was selected since it is the route of exposure, which is requested by regulatory authorities for this type of product.
- Rationale for animal assignment: random
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily at least once

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment and then once a week thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
male animals: first day of treatment, then once a week until sacrifice
female animals: first day of treatment, then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 1 and 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE :
The quantity of food consumed by each animal was recorded once a week, from the first day of each of the pre-mating, gestation and lactation periods. During the mating period, the food consumption was noted for neither males nor females .

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 males and 5 females of each group
- Parameters examined: Erythrocytes, Hemoglobin, Mean Cell Volume, Packed Cell Volume, Mean Cell Hemoglobin Concentration, Thrombocytes, Leucocytes, Differential White Cell count with morphology (neutrophils, eosinophils, basophils, lymphocytes, monocytes), Prothrombin Time, Activated Partial Thromboplastin Time, Fibrinogen.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the pre-mating period
- Animals fasted: Yes
- How many animals: 5 males and 5 females of each group
- Parameters examined: Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Total Bilirubin, Total Proteins, Albumin, Albumin/globulin ratio, Cholesterol, Triglycerides, Alkaline phosphatase, Aspartate aminotransferase, Alanine aminotransferase, Bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the pre-mating period, overnight, at least 14 hours
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Volume, pH, Specific gravity, Proteins, Glucose, Ketones, Bilirubin, Nitrites, Blood, Urobilinogen, Cytology of sediment (leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells), Appearance, Color


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: shortly before terminal sacrifice
- Dose groups that were examined: 5 male and 5 female animals of each group
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Dunnett, Fisher`s exact, Dunn, Mann-Whitney, Wilcoxon tests.
Details on results:
CLINICAL SIGNS AND MORTALITY
no substance related deaths, no substance related clinical signs observed.

BODY WEIGHT AND WEIGHT GAIN
1000 mg/kg bw/day - females: lower body weight gain in the females during gestation (minus 10% on days 0 - 20 of pregnancy as compared to the controls), and during lactation (minus 38% on days 1 to 4 post-partum). Males: no effect.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
1000 mg/kg bw/day - females: slightly lower food consumption in the females during gestation (minus 6% on days 0-20 of pregnancy).
Males: no effect.

HAEMATOLOGY
No notable changes.

CLINICAL CHEMISTRY
Females: Alanine aminotransferase increased at top-dose (18 as compared to 10 in controls; p<0.01) and slight increase of proteins in mid-dose, values within historical control range, and not considered as of biological significance. Males: significant, but not dose-related slight increases in Na, Cl, and Aspartate aminotransferase values. All values were within historical control ranges and not considered as of biological significance.

URINALYSIS
No notable changes.

NEUROBEHAVIOUR
No effect on motor activity and reflexes observed

ORGAN WEIGHTS
No substance related differences.

GROSS PATHOLOGY
No substance related pathological changes

HISTOPATHOLOGY: NON-NEOPLASTIC
Males: dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the 300 and 1,000 mg/kg bw/day groups. As no tubular degeneration/necrosis was observed in the kidneys the presence of acidophilic globules was considered as due to the accumulation of the sex-linked alpha-2-u-globulin. This was confirmed by Mallory-Heidenhain and specific immunostaining in a specific investigation. As a sex- and species-specific effect of male rats, this finding has no relevance for humans. It was therefore considered as of minor toxicological importance and it was not considered as an adverse effect. The seminiferous tubules were lined with Sertoli cells only (minimal or slight) in 1/10 males given 300 mg/kg/day and in 2/10 males given 1000 mg/kg/day. For a third male from the same group, tubules lined with Sertoli cells only were considered to be tubuli recti as they were situated beneath the capsule. For 1/10 males given 300 mg/kg/day and another given 1000 mg/kg/day, minimal reduction in the number of spermatids was observed in very few seminiferous tubules. Minimal vacuolization of Sertoli cells was observed in 1/10 males given 1000 mg/kg/day. Although not found in the control males, these microscopic abnormalities recorded with minimal severity in few or very few seminiferous tubules in a few males were considered to be without relationship to the treatment and most probably fortuitous.
In summary, no treatment-related abnormalities were found in testes, epididymides, prostate, seminal/vesicles, ovaries, and uterus, and in all other investigated organs.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: body weight; food consumption during pregnancy and lactation
Key result
Dose descriptor:
other: NOAEL not relevant for humans
Effect level:
100 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: histopathology: higher severity of acidophilic globules in the kindneys due to accumulation of alpha-2-u-globulin. As a sex-and species-specific effect of male rats, this finding has no relevance for humans
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: Highest dose, taking into account that alpha-2-u-globulin accumulation in male rats is not relevant for humans.
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and guideline study

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
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

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

The repeated dose toxicity of IBDU (purity 90.1 %) was assessed in a study on rats according to OECD guideline 422 (combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test); (BASF AG, 2003). 10 male and 10 female Sprague-Dawley rats per group were treated by gavage with 0, 100, 300 or 1000 mg IBDU/kg bw/day. Males were treated during pre-mating (15 days), mating (max. 14 days) and post-mating for a total of 34 days, and females during pre-mating (15 days), mating (max. 14 days), and pregnancy and lactation until day 4 post partum.

There were no substance related deaths, nor were there any substance related clinical signs observed. The high-dose females showed lower body weight gain during gestation and during lactation. They also had slightly lower food consumption during gestation. No effects on body weight gain or food consumption were seen in males up to and including the highest tested dose.

The treatment with IBDU had no effects on hematology, blood biochemistry and urine parameters.

There were no substance related pathological changes found at necropsy. In males, the histopathological examination revealed a dose-related higher severity of acidophilic globules in the cortical tubular epithelium of the kidneys of the 300 and 1000 mg/kg bw/day groups. As no tubular degeneration/necrosis was observed in the kidneys, the presence of acidophilic globules was considered as due to the accumulation of the sex-linked α-2-u-globulin. This was confirmed by Mallory-Heidenhain and specific immunostaining (BASF, 2004). As a sex- and species-specific effect of male rats, this finding has no relevance for humans. It was therefore considered of minor toxicological importance and not considered as an adverse effect. No treatment-related abnormalities were found in testes, epididymides, prostate, seminal/vesicles, ovaries, and uterus, and in all other investigated organs.

The “No Observed Effect Levels” (NOAELs) were 300 mg/kg bw/day for females (reduced body weight gain during pregnancy and lactation at 1000 mg/kg bw/day), and 1000 mg/kg bw/day for males.

Further, the repeated dose toxicity of IBDU was assessed in a study according to OECD guideline 408 (BASF SE, 2017). The test substance was administered by gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0, 100, 300 and 1000 mg IBDU/kg bw/day over a period of 3 months. Drinking water containing 0.5% Carboxymethylcellulose served as vehicle.
No treatment-related, adverse effects were observed at low and mid dose group of 100 and 300 mg/kg bw/day. At 1000 mg/kg bw/day increased protein storage in tubules (eosinophilic droplets, positive CAB staining), increased incidence of basophilic tubules as compared to controls, and tubular casts in male animals (α2u-nephropathy) were observed.

The oral administration of the test substance by gavage to male and female Wistar rats for 3 months caused adverse signs of toxicity in male animals, only, paying attention to the tubular damage in the kidneys at 1000 mg/kg bw/d. However, the no observed adverse effect level (NOAEL) for general systemic toxicity was set to 1000 mg/kg bw/d for male and female Wistar rats taking into account that tubular damage in the kidneys of male Wistar rats were a consequence of an α-nephropathy which does not represent a risk for humans.

 

Repeated dose toxicity: inhalation

This information is not available

 

Repeated dose toxicity: dermal

This information is not available

Justification for classification or non-classification

After repeated oral administration over 4 weeks by gavage to rats in a screening study following OECD guideline 422 , the “No Observed Effect Levels” (NOAELs) were 300 mg/kg bw/day for females (reduced body weight gain during pregnancy and lactation at 1000 mg/kg bw/day), and 1000 mg/kg bw/day for males (highest tested dose level). In an oral repeated dose study in rats according to OECD guideline 408 the NOAEL for general systemic toxicity was 1000 mg/kg bw/d for male and female rats. As no other substance related findings were made which were relevant to human health, no classification and labeling is required concerning repeated exposure to IBDU.