Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2008 - October 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
caging of the Wistar rats at the test facility started before the study inition date
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
none

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Species: Rattus norvegicus
Strain: Wistar-Unilever
Sex: 20 male, 20 female
Source: Harlan Winkelmann GmbH
Gartenstraße 27
33178 Borchen, Germany
Total number of animals: 40
Age at experimental starting date: 5 – 8 weeks
Health Status: specific pathogen free (SPF)
Caging: 8 groups of five animals each in open makrolon cages type
2000P (TechniPlast) Bedding Lignocel hygienic animal bedding (J. Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg)
Diet Maintenance diet rat/mouse, pellets, No. 1324 (Altromin GmbH & Co. KG, 32791 Lage), ad lib.
Water Autoclaved community tap water, ad lib.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room climate 22 ± 3°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): housing: aeration with approx. 10 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial lighting 12 h light/12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
Maintenance diet rat/mouse, pellets, No. 1324 (Altromin GmbH & Co. KG, 32791 Lage), ad lib.


VEHICLE
- Justification for use and choice of vehicle (if other than water): As the test item’s solubility in water is poor, corn oil was used as an organic solvent. Corn oil
is very well investigated as vehicle for toxicity studies, and it is explicitly recommended by the relevant guideline OECD 407.
- Concentration in vehicle: After the required amount of the test substance was weighted into a dispersion tube, corn oil was added up to the required volume (appication volume: 8 ml)
- Amount of vehicle (if gavage):
- Lot/batch no. (if required): 126K0117 (Sigma, Catalogue number C8267)
- Purity: 100%
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily administration
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
63 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:

No. of animals per sex per dose:
5 male: dose levels of 63, 250 and 1000 mg/kg BM/day
5 female dose levels of 63, 250 and 1000 mg/kg BM/day
Control animals:
other: yes; same total volume of water as vehicle control
Details on study design:
- Dose selection rationale: In a previously performed dose range finding study with dose escalation2, the test item was administered in doses up to 2000 mg/kg body mass over a time period of 19 days and produced no observable toxic effects in the test animals. In accordance with the sponsor, 1000 mg/kg was determined as high dose for this test.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first application, and once a week thereafter.
- changes in skin, fur, eyes, mucous membranes
- occurrence of secretions and excretions
- autonomic activity (e.g. lacrimation, piloerection, unusual respiratory patterns)
- changes in gait, posture, and response to handling
- presence of clonic or tonic movements, stereotypies (e.g. excessive grooming,
repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards)
- and any other observed, unusual signs

GRIP STRENGTH AND LIMB PLACING TEST:
For the grip strength analysis, rats gripping the cage top were pulled off by a spring balance attached to their tail. The maximum read-out of the balance showed the strength of the rat’s hold on the cage bars. On day one and in the last exposure week, additional recordings were made of grip strength and reactivity to stimuli (limb placing test [De Ryck et al. (1989), modified by Jolkkonen et al. (2000)])

BODY MASS: Yes
- Time schedule for examinations: once weekly

FOOD AND WATER CONSUMPTION: Yes
- Group food consumption Once weekly (including once before application start)
- Group water consumption Twice weekly (including once before application start)

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

Blood sampling, hematology and serum biochemistry:
On day 29, approx. 200 – 550 μl blood3 was taken from the retroorbital vein plexus of each animal immediately before sacrification. In order to avoid coagulation, 90 μl of each individual blood sample was mixed with 10 μl 10x HEPES-EDTA solution directly after bleeding. 30 μl of the remaining blood was used to directly determine the blood clotting time and potential, the rest of the blood was prepared as serum. Blood and serum samples were transferred to the Principal Investigator 1 to analyse parameters on hematology and serum biochemistry (see table 4) within 8 hours after bleeding. The original results of the analysis were returned to the hands of the study director at NewLab BioQuality GmbH.

Sacrifice and pathology:
The in life phase was terminated on day 29. Animals were sacrificed by asphyxiation in a CO2 atmosphere.

GROSS PATHOLOGY: Yes (see table 1)
A full macroscopic examination was performed and all occurring lesions were noted. Individual animal data were recorded on checklists. The weight of the organs denoted by „W“ in table 1 was recorded; tissues denoted by „P“ were preserved in the appropriate fixatives.


HISTOPATHOLOGY: Yes (see table 1)
Histological preparation was performed of the organs and tissues denoted by „P“ in table 2. The selected tissues were embedded in paraffin wax, sectioned, and stained with hemalaum and eosin. The stained sections were transferred to the Principal Investigator 1 for histopathological examination.
Other examinations:
Hematology and serum biochemistry

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No abnormalities concerning general clinical signs or behaviour were observed.
Mortality:
no mortality observed
Description (incidence):
No abnormalities concerning general clinical signs or behaviour were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
weight increase within normal range for rats of this strain and age
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
food consumption of all animals was within normal range for rats of this strain and age
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
within normal range for rats of this strain and age
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Analysis of haematology and serum biochemistry showed normal results in most instances. Although some significant changes were observed, none of the observed average data points were overall extremely out of range for rats of this strain and age
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Analysis of haematology and serum biochemistry showed normal results in most instances. Although some significant changes were observed, none of the observed average data points were overall extremely out of range for rats of this strain and ag
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
relative organ mass of the kidneys of the male test animals of the high dose group was slightly but significantly increased. The relative organ mass of the right adrenal gland of the female animals of the high dose group was mildly but significantly raise
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
morphological lesions that were considered to be related to the test item
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormalities concerning general clinical signs or behaviour were observed.
No animal of the test item groups or the vehicle groups died during the in-life phase of this study.
In the course of this study, no signs of illness (e.g. changes in skin, secretions), autonomic activity (e.g. lacrimation, piloerection), stereotypies or behavioural reactions (e.g. selfmutilation) in response to the treatment or otherwise was observed.

BODY WEIGHT AND WEIGHT GAIN
within normal range for rats of this strain and age. Male rats subjected to the test item gained slightly less weight than their vehicle control group, but, as all other animal groups treated with the test item, showed no significant alterations of their body mass when compared to their control groups

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In comparison with the vehicle control group, the average food uptake of the female high dose group was slightly raised, and the average food uptake of the male medium dose group was slightly lowered during in-life phase. However, the food consumption of all animals was within normal range for rats of this strain and age

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The water consumption of all animals was within normal range for rats of this strain and age. Water consumption of the male high dose group was raised during the last week, but only due to a high consumption during the first half of week four, in the second half of week four
water consumption was back to normal. Based on this observation, it can be assumed that this peak water consumption does not reflect a
higher water intake, but was caused by a minor bottle leakage or by the test animals playing with the drinking water.

Grip strength and Limb placing test:
Motor activity and reactivity to visual and proprioceptive stimuli were not altered by the administration of the test item. At the end of the observation period, no differences in grip strength were observed in relation to the vehicle control group, and none of the animals showed significant differences to the vehicle group during the limb placing test.

ORGAN WEIGHTS
relative organ mass of the kidneys of the male test animals of the high dose group was slightly but significantly increased. The relative organ mass of the right adrenal gland of the female animals of the high dose group was mildly but significantly raise (see also Necropsy/Gross Pathology).

GROSS PATHOLOGY:
In comparison to their control group, the relative organ mass of the kidneys of the male test animals of the high dose group was slightly but significantly increased. The relative organ mass of the right adrenal gland of the female animals of the high dose group was mildly but significantly raised. Data for the left adrenal gland of the female high dose group showed the same tendency, but did not reach the significance niveau.

HISTOPATHOLOGY:
seminal vesicle in both control and/or test item treated animals. The inflammatory reaction was associated with minimal to mild lymphoid hyperplasia in the spleen, lymph nodes and the gut-associated lymphoid tissue in the intestine. A minimal reduction of lymphoid tissue (involution) was noted in the cortex and medulla of the thymus in the male and female rats of all groups.
Coincidental findings in a small number of control and/or test item-treated animals are:
Epididymis: immature spermatozoa in the lumen;
Larynx: glandular ectasia;
Liver: extra medullary haematopoiesis;
Ovary: follicular cysts;
Testis: atrophy of germinative epithelium;
Trachea: tracheal gland dilatation;
Urinary bladder: proteinaceous content in male rats, dilatation;
Uterus: hydrometra.
The histomorphological examination of rat organs from the 28-day toxicity study by oral administration of animals treated with IPBC did not reveal morphological lesions that were considered to be related to the test item. There was no morphological difference between the vehicle control group and the groups subjected to the high dose of the test item.

Effect levels

Dose descriptor:
dose level:
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

Executive summary:

A daily oral administration of the test item to Wistar rats at a dose level of 63, 250 and 1000 mg/kg body mass over a time period of 28 days resulted in only mild systemic and local effects in test animals treated with the high dose (1000 mg /kg) but did not produce any pathological evidence of a local or systemic toxicity of the test item.