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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N''-hexane-1,6-diylbis[N'-benzylurea]
EC Number:
254-274-3
EC Name:
N,N''-hexane-1,6-diylbis[N'-benzylurea]
Cas Number:
39072-70-3
Molecular formula:
C22H30N4O2
IUPAC Name:
3-benzyl-1-{6-[(benzylcarbamoyl)amino]hexyl}urea
Details on test material:
- Test Item: N,N“-Hexane-1,6-diylbis[N`-benzylurea]
- BASF Test Item No.: 11/0726-1
- Batch Number: 259-635
- Purity: 98.0 g/100 g determined by 1H NMR spectroscopy using the internal standard method
- Physical state, appearance: White solid
- Homogeneity: The test substance appeared to be homogeneous.
- Storage conditions: At room temperature, no direct sunlight, protect against humidity

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland
- Age at study initiation: Approximately 11 weeks
- Weight at study initiation: body weight of all animals within ± 20% of the sex mean.
- Housing:
Pre-mating:
Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating:
Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating:
Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation:
Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 room air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test substance.
VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (27 August 2012), according to a validated method (Project 499988, BASF Project 05Y0726/11X510). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-53 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 44 (Group 1) 53, 56 (Group 2), 71 and 72 (Group 4) were not dosed during littering.
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the 14-Day dose range finding study
- Rationale for animal assignment (if not random): Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
N/A

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
At least twice daily for mortality. Daily, detailed clinical observations were made in all animals, at least immediately (0-15 min) after dosing. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. Since no clinical observations were noted in the dose range finding study, observations were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

WATER CONSUMPTION: Yes
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

CLINICLA LABORATORY INVESTIGATIONS:
Blood samples were collected from the selected 5 animals/sex/group (see Allocation) under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.

The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin (0.5 mL) for clinical biochemistry parameters. An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids (tubes: Greiner Bio-One GmbH, Kremsmünster, Austria).

Furthermore, an additional blood sample (0.5 mL) was collected from the 5 selected animals/sex/group (see Allocation) into serum tubes for possible future measurement of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, T3 and T4 were stable for 2 months. Any samples remaining at finalization of the study report were discarded. The T3 and T4 investigations were only conducted if necessary.

HAEMATOLOGY: Yes
The following haematology parameters were determined in blood prepared with EDTA as an anti-coagulant, using the ADVIA® 120 Hematology System (Siemens Healthcare Diagnostics B.V., Breda, The Netherlands):
White blood cells (WBC)
Differential leucocyte count: Neutrophils, Lymphocytes, Monocytes, Eosinophils and Basophils
Red blood cells, Reticulocytes, Red blood cell distribution width (RDW), Haemoglobin, Haematocrit, Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelets
The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France): Prothrombin Time (PT) and Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
The following clinical biochemistry parameters were determined using the AU400® Chemistry System (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum:
Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline Phosphatase (ALP), Total protein, Albumin, Total Bilirubin, Bile acids, Urea, Creatinine, Glucose, Cholesterol, Sodium, Potassium, Chloride, Calciumand Inorganic Phosphate (Inorg. Phos)

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observations
The following tests were performed on the selected 5 animals/sex/group (see Allocation):
hearing ability, pupillary reflex, static righting reflex, grip strength and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

During motor activity monitoring, all animals were caged individually (pups were housed in their home cages and kept warm using bottles filled with warm water).

The selected males were tested during Week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and before blood sampling.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All males and the selected 5 females/group were deprived of food overnight, the evening before the scheduled necropsy (with a maximum of 24 hours), but water was provided. Non-selected females were not deprived of food.
Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Females which delivered on Lactation Days 5-7.
The female which failed to deliver (no. 73) on Post-coitum Day 26 (female with evidence of mating)
Female with litter loss (no. 55) within 24 hours.
Males: Following completion of the mating period (a minimum of 28 days of dose administration).

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.

The numbers of former implantation sites and corpora lutea were recorded for all paired females. These numbers were not reported for non-pregnant and non-mated females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).

Samples of the following tissues and organs were collected from all animals and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Ovaries, Adrenal glands, (Pancreas), (Aorta), Peyer's patches [jejunum, ileum] if detectable, Brain - cerebellum, mid-brain, cortex, Pituitary gland, Caecum, Preputial gland, Cervix, Prostate gland, Clitoral gland, Rectum, Colon, (Salivary glands - mandibular, sublingual), Coagulation gland, Sciatic nerve Duodenum, Seminal vesicles, Epididymides[1], Skeletal muscle, Eyes (with optic nerve (if detectable) and Harderian gland)[1], (Skin), Spinal cord -cervical, midthoracic, lumbar, (Male and female mammary gland area), Spleen, Femur including joint, Sternum with bone marrow, Heart, Stomach (forestomach and glandular stomach), Ileum, Testes[1], Jejunumm, Thymus, Kidneys, Thyroid including parathyroid if detectable, (Lacrimal gland, exorbital), (Tongue), (Larynx), Trachea, Liver, Urinary bladder, Lung, infused with formalin, Uterus, Lymph nodes - mandibular, mesenteric, Vagina, (Nasopharynx), All gross lesions, (Esophagus)

[1]: Fixed in modified Davidson's solution (prepared at WIL Research Europe using Formaldehyde 37-40%, Ethanol, Acetic acid (glacial) (all Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)) and transferred to formalin after fixation for at least 24 hours.

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

HISTOPATHOLOGY: Yes
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

Of all animals of the control and high dose group and all males suspected to be infertile, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
A peer review on the histopathology data was performed by a second pathologist.

The following slides were examined by a pathologist:
- The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
- The additional slides of the testes of all males of Groups 1 and 4 and all males suspected to be infertile to examine staging of spermatogenesis.
- All gross lesions of all animals (all dose groups).
- The reproductive organs[*] of all animals of Group 1 and 4 and of male no. 15 and 33 (failed to sire) and female no. 55 (litter loss) and no. 73 (no offspring).
[*] Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.

Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.
Other examinations:
Organ Weights:
The following organ weights and terminal body weight were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands, Spleen, Brain, Testes, Epididymides, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate[2], Liver, Seminal vesicles including coagulating glands[2], Ovaries, Thyroid including parathyroid[2],
[2] weighed when fixed for at least 24 hours.
All remaining males:
Epididymides and Testes
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.

The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

One female (no. 55) was found littering on 26 September 2012 (post-coitum Day 22), but no pups were found on 27 September 2012. This animal was therefore sent for unscheduled necropsy due to “total litter loss”. At necropsy, one pup was found left in the uterus.

No toxicologically relevant clinical signs were noted.

Pale faeces noted at 1000 mg/kg bw/day in both sexes from week 1 of treatment onwards, was attributed to the white colour of the test substance, and considered not to be of toxicological relevance.

Salivation seen after dosing for most males at 1000 mg/kg bw/day during the last week of treatment was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.
Salivation was also incidentally noted for one female at 300 mg/kg bw/day. Together with other incidental findings including rales, alopecia, green discolouration of the urine and scabs, these were considered signs of no toxicological relevance. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically relevant changes in body weights and body weight gain were noted.

The statistically significant higher body weight of females at 1000 mg/kg bw/day on Day 8 of the premating period (including statistically significant higher body weight gain) and on most occasions during post-coitum/lactation remained within the range considered normal for rats of this age and strain, and showed no clear dose-related trend over the dose groups. No toxicological relevance was therefore ascribed to these changes.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

Any statistically significant changes in food consumption of females during post-coitum/lactation remained within the range considered normal for rats of this age and strain and occurred in the absence of a clear dose-related trend over the dose groups. No toxicological relevance was therefore ascribed to these changes.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters of treated rats.

The statistically significant lower prothrombin time (PT) in males at 100 and 300 mg/kg bw/day, and higher relative basophil counts in females at 1000 mg/kg bw/day were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.

NEUROBEHAVIOUR
No toxicologically relevant changes in motor activity were noted. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

Mean total movements of females at 1000 mg/kg bw/day were higher than controls at the end of the lactation period. There were no concurrent clinical signs, and all groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period. This change was therefore considered not to be adverse in nature. The variation in motor activity at 100 and 300 mg/kg bw/day did not indicate a relation with treatment.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.

The statistically significant higher liver weight and liver to body weight ratio of females at 300 mg/kg bw/day occurred in the absence of a dose-related trend and was therefore considered to be of no toxicological relevance.


GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations.

The incidence of macroscopic findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show a dose-related incidence trend, and occurred in the absence of treatment-related histopathological changes. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY
All microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar-Han rats of this age.

No histopathological changes were noted in the reproductive organs of two Group 2 animals (male no. 15 and female no. 55) and two Group 4 animals (male no. 33 and female no. 73) that could account for failing to deliver healthy offspring.

The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item.

Effect levels

Dose descriptor:
NOAEL
Remarks:
local and systemic
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

N,N”-Hexane-1,6-diylbis[N’-benzylurea] was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for 2 weeks prior to mating, during mating, and up to termination (for 29 days). The females were exposed for 2 weeks prior to mating, during mating, duringpost-coitum, and at least 4 days of lactation (for 42-53 days).

 

Formulation analysis showed that the formulations were prepared accurately and homogenously, and were stable for at least 6 hours at room temperature.

Parental results:

No parental toxicity was observed up to the highest dose level tested (1000 mg/kgbw/day).

 

No toxicologically significant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

 

Reproductive results:

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kgbw/day).

 

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

 

Developmental results:

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kgbw/day).

 

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e.gestation index and duration, parturition, maternal care and earlypostnatalpup development consisting of mortality, clinical signs, body weight and macroscopy).

 

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg bw/day was derived.

Applicant's summary and conclusion