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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Details on test material:
- Name of test material (as cited in study report): Hexane, 1,6-diisocyanato, homopolymer, N-butyl-1-butanamine-blocked
- Test-substance No.: 11/0689-1
- Lot/batch No.: IB 60/0053
- Purity: approx. 100 %
- Homogeneity: The test item was homogeneous by visual inspection
- Storage stability: stable
- Storage conditions: At room temperature in the dark
- Physical state/ colour: Solid / colourless to yellowish

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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. The test substance was warmed before weighing (maximum temperature=66.9°C, maximum duration=6 hours) and the formulations were also warmed to facilitate mixing (maximum temperature=69°C, maximum duration=57 minutes). 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:
Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at ABL, The Netherlands, where samples were stored at ≤-70°C until analysis.

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% 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 42-56 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).
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. 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. 54) on Post-coitum Day 27 (female with evidence of mating)
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. 14 (failed to sire) and female no. 54 (failed to deliver healthy pups).
[*] 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. No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included alopecia, scabs on the neck and piloerection. A single female at 300 mg/kg bw/day had a wound and swelling along with dull appearance and opacity of the left eye. 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. At the incidence observed, these were not considered treatment related or toxicologically relevant.

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

Absolute body weights were significantly lower on Day 1 of the mating period and Day 4 of the lactation period for females at 300 and 100 mg/kg bw/day, respectively. In the absence of a treatment related distribution, this was not considered to be toxicologically relevant.

FOOD CONSUMPTION
No toxicologically relevant changes in food consumption before or after allowance for body weight were noted.

Absolute food consumption was significantly lower for females at 100 mg/kg bw/day on Days 11-14 of the post coitum period and Days 1-4 of the lactation period. Food consumption was also significantly lower for females at 300 mg/kg bw/day on Days 17-20 of the post coitum period. As the difference from controls was only slight and occurred in the absence of a treatment related distribution, it was not considered toxicologically relevant.

HAEMATOLOGY
There were no differences noted in haematological parameters between control and treated rats that were considered to be related to treatment with Hexane, 1,6-diisocyanato, homopolymer, N-butyl-1-butanamine-blocked.

The statistically significant decrease in haemoglobin seen for females at 300 mg/kg bw/day was not considered to be toxicologically relevant as it occurred in the absence of a treatment-related distribution.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.

Urea was significantly increased for females at 300 mg/kg bw/day. No toxicological relevance was attributed to this difference as there was no dose-dependent effect noted.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

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

Relative liver weights were significantly lower for males at 1000 mg/kg bw/day. This was not toxicologically relevant as an increase in liver to body weight ratios would be expected if treatment related toxicity was evident. Similarly, the significantly lower terminal body weight seen for females at 100 mg/kg bw/day was not considered toxicologically relevant.

GROSS PATHOLOGY
Macroscopic observations at necropsy did not reveal any alterations that were attributable to treatment.

All macroscopic findings noted were incidental in nature. These included pelvic dilation of the kidneys, tan, red-brown or reddish discoloration of the thymus and mandibular lymph node, reduced size of the left prostate, several tan foci on the right clitoral gland, thickened limiting ridge of the stomach, enlarged spleen, mesenteric lymph node and/or liver, cloudy left eye, yellowish, hard nodule on the uterine adipose tissue, scab on the back of the neck and alopecia on the throat, flank, or foreleg. These findings remained within the range of findings encountered among rats of this age and strain, and none were attributable to treatment. None of these were considered to be toxicologically relevant.

HISTOPATHOLOGY:
There were no treatment-related microscopic findings. All recorded microscopic findings were within the normal range of background alterations encountered in Wistar (Han) rats of this age and strain.

No abnormalities were seen in the reproductive organs of the rats who failed to sire or deliver healthy pups, which could account for their infertility.

Furthermore, the spermatogenic staging profiles were normal for all Group 1 and 4 males, and for all males suspected to be infertile.

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

Applicant's summary and conclusion