N,N''-(isobutylidene)diurea

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

21 Sep 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Remarks:

BASF SE, Experimental Toxicity and Ecology, 67056 Ludwigshafen, Germany.

Test material

Test material information

Constituent 1

Reference substance name:

N,N''-(isobutylidene)diurea

EC Number:

228-055-8

EC Name:

N,N''-(isobutylidene)diurea

Cas Number:

6104-30-9

Molecular formula:

C6H14N4O2

IUPAC Name:

N,N''-(2-methylpropane-1,1-diyl)diurea

Test material form:

solid: crystalline

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 2015-03-24

Test animals

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 or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

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

Results and discussion

Results of examinations

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)

Effect levels

1

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.

2

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)

3

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

Target system / organ 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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.