Registration Dossier

Administrative data

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-reference
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
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
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Wistar Han ™
Sub-strain: RccHan™:WIST
Source of supply: Envigo RMS (UK) Ltd., Oxon, UK
Number: 80
Sex: Males and females
Age: Approximately 6 to 8 weeks at start of treatment
Other: Weight variation did not exceed ± 20% of the mean weight for either sex at start of treatment
Sex:
male/female
Details on test animals or test system and environmental conditions:
Temperature: 22 ± 3 °C
Humidity: 30 to 70%
Lighting: Twelve hours of continuous artificial light in each twenty-four hour period
Ventilation: At least fifteen air changes per hour

Animals were housed in accordance with the 'Code of Practice for the Housing and Care of Animals Used in Scientific Procedures' and in alignment with the United Kingdom Home Office recommendations and requirements.
Route of administration:
oral: gavage
Details on route of administration:
Once daily, by gavage, using a stainless steel dosing cannula attached to a graduated syringe for ninety consecutive days. Dosing was performed at a similar time each day wherever possible.
Vehicle:
polyethylene glycol
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once per day
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
NA
Observations and examinations performed and frequency:
1.1 Morbidity/Mortality Inspection
Twice daily, early and late during the working period.

1.2 Clinical Observations
Individual clinical observations were performed immediately before dosing, up to 30 minutes after dosing and one hour after dosing.

1.3 Body Weights
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Individual body weights were also be recorded at terminal kill.

1.4 Food Consnmption
Dietary intake was recorded weekly for each cage group. Weekly food efficiency (body weight gain/food intake) was calculated.

1.5 Water Consumption
Water consumption was monitored daily by visual inspection of water bottles.

2 Specialist Evaluations
2.1 Functional Observations
Detailed individual clinical observations were performed on all test and control group animals before the first exposure to the test item and once weekly thereafter. These observations were performed outside the home cage, in a standard arena, at least two hours after dosing (where applicable) to ensure that any transient effects of treatment were identified. In addition, sensory reactivity to different stimuli (auditory, visual and proprioceptive), grip strength (fore and hind limb) and motor activity (using a 44 photobeam unit) were measured once during Week 12 of the study, at least two hours after dosing.

2.2 Ophthalmoscopy
Ophthalmoscopic examination was performed on all animals before the start of treatment and all control and high dose animals during Week 12 of the study. Examinations included observation of the anterior structures of the eye and, following pupil dilation, detailed examination of the internal structures of the eye using a ophthalmoscope.

3 In-life Sampling and Analysis
Hematological and blood chemical investigations were performed on all test and control group animals at the end of the treatment period (Day 90). Blood samples were withdrawn from the lateral tail vein. Repeat blood samples were withdrawn by cardiac puncture at termination if required. Animals were fasted prior to blood sampling.

3.1 Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Prothrombin time (CT)
Platelet count (PLT)
Activated partial thromboplastin time (APTT)
Reticulocyte count (Retic)

3.2 Blood Chemistry
Urea
Glucose
Total protein (Tot.Prot.)
Albumin
Albumin/Globulin (A/G) ratio (by calculation)
Sodium (Na+)
Potassium (K +)
Chloride (Cl-)
Calcium (Ca++)
Inorganic phosphorus (P)
Aspartate aminotransferase (ASAT)
Alanine aminotransferase (ALAT)
Alkaline phosphatase (AP)
Creatinine (Creat)
Total cholesterol (Chol)
Total bilirubin (Bili)
Bile acids
Sacrifice and pathology:
1 Necropsy
Post mortem procedures were performed on all animals including the animals found dead or killed in extremis during the study. All animals were killed by
intravenous overdose of a suitable barbiturate agent, followed by exsanguination at termination. Selected tissues were weighed and whole or samples of required tissues were retained in 10% Buffered Formalin (unless otherwise indicated). The lungs were inflated to approximately normal respiratory volume with buffered 10% formalin before immersion in fixative.

Tissue/Organ Weigh Retain Process &
Examine
Adrenals X X X
Aorta (thoracic) X X
Bone and bone marrow (femur including stifle joint) X
Bone and Bone marrow (sternum) X X
Brain (including cerebrum, cerebellum and pons) X X X
Caecum X X
Colon X X
Duodenum X X
Esophagus X X
Epididymides (retained in Modified Davidson's X X X
Fluid)
Eyes (retained in Davidson's Fluid) X X
Gross lesions X X
Heart X X X
Ileum (including peyer's patches) X X
Jejunum X X
Kidneys X X X
Liver X X X
Lungs (with bronchi) X X
Lymph nodes (mandibular and mesenteric) X X
Mammary gland X X
Muscle (skeletal) X
Ovaries X X X
Pancreas X X
Pituitary X X
Prostate X X
Rectum X X
Salivary glands (submaxillary) X X
Sciatic nerve X X
Seminal vesicles (including coagulating gland) X X
Skin X X
Spinal cord (cervical, mid-thoracic and lumbar) X X
Spleen X X X
Stomach X X
Testes (retained in Modified Davidson's Fluid) X X X
Thymus X X X
ThyroidlParathyroid X X
Tongue X
Trachea X X
Urinary bladder X X
Uterus (with Cervix) X X X
Vagina X X

1 Gross Examination
Full external and internal macroscopic examination of all animals.

1.2 Organ Weights
The tissues listed in the table in Section 1 were weighed for all surviving animals at terminal kill. As far as was possible, fat and connective tissues were trimmed from each organ and the organs were weighed fresh prior to being placed in fixative. Generally paired organs were weighed together and not separately.

2 Histology
Samples of the tissues preserved in their appropriate fixatives were dispatched to the histology processing Test Site.
Initially:
a) all tissues from animals that die or are killed in extremis during the study
b) all tissues from control and high dose group animals
c) any abnormal tissues from low and intermediate dose groups were routinely processed to paraffin wax, sectioned and stained with Haematoxylin and
Eosin. In addition, sections of testes were also stained with Periodic Acid-Schiff (PAS) stain. The examination of gross lesions is intended to cover the normal background incidence of macroscopic gross lesions observed for this study design.

3 Pathology
3.1 Histopathology
Prepared slides were sent to the Study Pathologist, whereupon special staining techniques may be used, where appropriate.
Suspected treatment-related lesions were seen in the high dose group, therefore histology processing and histopathological examination of the affected tissue(s) were extended to animals of the low and intermediate dose groups.
Statistics:
Statistical Analysis
Quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was
achieved at a level of p<0.05. Analysis was performed on, but not necessarily restricted to, the following parameters:
Body weight change, motor activity, grip strength, hematology, blood chemistry, absolute organ weights and body weight-relative organ weights
Data was analyzed using the decision tree from the Provantis ™ Tables and Statistics Module as detailed below:
Transformation of the data was performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett's test.
Intergroup variance was assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data was analyzed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows nonhomogeneity of means, the data was analyzed by a stepwise Dunnett's (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data is unsuitable for these analyses, then pair-wise tests was performed using the Student t-test (parametric) or the Marm-Whitney U test (non-parametric).
Description (incidence and severity):
Increased salivation was evident in animals of either sex treated with 600 and 100 mg/kg bw/day throughout the treatment period. No such effects were detected in animals of
either sex treated with 30 mg/kg bw/day.
Description (incidence):
There have been no treatment-related deaths.
One female treated with 30 mg/kg bw/day was killed in extremis on Day 75 due to adverse clinical signs (pilo-erection, hunched posture, pallor of the extremities, stained snout and hypothermia). In the absence of any similar clinical signs evident in 100 or 600 mg/kg bw/day animals, this death was considered to be incidental.
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter. No such effects were detected in
females treated with 600 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.
Description (incidence and severity):
No overall adverse effect on food consumption was evident.
Description (incidence and severity):
Visual inspection of water bottles did not revealed any inter-group differences.
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed statistically significant reductions in hemoglobin and mean corpuscular hemoglobin concentration and a statistically significant increase in neutrophils. The effect on mean corpuscular hemoglobin concentration also extended to males treated with 100 mg/kg bw/day. Males treated with 600 mg/kg bw/day showed a statistically significant reduction in activated partial thromboplastin time. Females treated with 600 mg/kg bw/day showed a statistically significant reduction in mean
corpuscular hemoglobin concentration and a statistically significant increase in neutrophils. The effect on neutrophils also extended to females treated with 100 mg/kg bw/day.
No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured.
Description (incidence and severity):
Behavioural Observations.
There were no inter-group differences considered to be treatment-related.

Functional Performance.
There were no treatment related changes in functional performance considered to be related to treatment at 30, 100 or 600 mg/kg bw/day.

Sensory Reactivity.
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 30, 100 or 600 mg/kg bw/day.
Description (incidence and severity):
No toxicologically significant effects were detected in the organ weights measured.
Description (incidence and severity):
Six males treated with 600 mg/kg bw/day had sloughing on the non-glandular region in the stomach. Two of these males also had a raised limiting ridge and one also had a raised limiting ridge and a thickened stomach. A further male had a thickened stomach and a raised limiting ridge and another male had a raised limiting ridge. Two females treated with 600 mg/kg bw/day had a raised limiting ridge and one of these females also had a thickened stomach whilst the other also had gaseous distension in the caecum. A further female had sloughing on the non-glandular region of the stomach. One male treated with 100 mg/kg bw/day had a raised limiting ridge in the stomach and one female treated with 30 mg/kg bw/day had a raised limiting ridge in the stomach, thickening of the nonglandular region of the stomach, sloughing in the stomach and reddened lungs. The female treated with 30 mg/kg bw/day that was killed in extremis on Day 75 had dark red contents
in the stomach and intestines.
Description (incidence and severity):
The following treatment related microscopic abnormalities were detected:

Stomach
Hyperplasia in the non-glandular region in males and females (minimal to moderate) Erosion of the glandular mucosa generally close to the limiting ridge (minimal or mild) Increased inflammatory cell infiltration in the glandular region (usually associated with erosion, minimal) Hyperplasia of the glandular mucosa females only (localised, foveolar, minimal)

Duodenum
Mucosal hypertrophy in males and females (minimal or mild)
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No relevant systemic toxicity
Critical effects observed:
not specified
Conclusions:
Based on the results of this study, the NOAEL was 600 mg/kg bw/day for both sexes. There were some findings on the study were either sporadic and did not reflect an association with the dose of test substance, or were secondary effects caused by the irritant properties of the test substance.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guideline:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 30, 100 and 600 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Polyethylene glycol 400). Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals. All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

There were no treatment-related deaths. One female treated with 30 mg/kg bw/day was killed in extremis on Day 75 due to adverse clinical signs (pilo-erection, hunched posture, pallor of the extremities, noisy respiration, stained snout and hypothermia). At necropsy, this female had dark red contents in the stomach and intestines. In the absence of any premature deaths evident at 100 or 600 mg/kg bw/day, this death was considered to be incidental.

Clinical Observations

Increased salivation was evident in animals of either sex treated with 600 and 100 mg/kg bw/day during the treatment period. Incidences of noisy respiration were evident in animals of either sex treated with 600 mg/kg bw/day during the treatment period and an isolated incident of noisy respiration was evident in two males treated with 100 mg/kg bw/day and in one surviving female treated with 30 mg/kg bw/day. A single occurrence of red/brown staining around the snout was also evident in one male treated with 600 mg/kg bw/day. No such effects were detected in males treated with 30 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters at 30, 100 or 600 mg/kg bw/day.

Functional Performance Tests

There were no treatment-related changes in functional performance considered to be related to treatment at 30, 100 or 600 mg/kg bw/day.

Sensory Reactivity Assessments

There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 30, 100 or 600 mg/kg bw/day.

Body Weight

Males treated with 600 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter. No such effects were detected in females treated with 600 mg/kg bw/day or in animals of either sex treated with 100 or 30 mg/kg bw/day.

Food Consumption

No adverse effect on food consumption or food conversion efficiency was evident in treated animals.

Water Consumption

No adverse effect on water consumption was evident in treated animals.

Ophthalmoscopy

Ophthalmic examination of the eyes from rats treated with 600 mg/kg bw/day did not indicate any effect of treatment.

Hematology

Animals of either sex treated with 600 mg/kg bw/day and females treated with 100 mg/kg bw/day showed a statistically significant increase in neutrophil count. No such effects were detected in males treated with 100 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters measured.

Necropsy

Six males treated with 600 mg/kg bw/day had sloughing on the non-glandular region in the stomach. Two of these males also had a raised limiting ridge and one also had a raised limiting ridge and a thickened non-glandular region of the stomach. A further male had a thickened non-glandular region of the stomach and a raised limiting ridge and another male had a raised limiting ridge. Two females treated with 600 mg/kg bw/day had a raised limiting ridge and one of these females also had a thickened non-glandular region of the stomach whilst the other female also had gaseous distension in the caecum. A further female had sloughing on the non-glandular region of the stomach. One male treated with 100 mg/kg bw/day had a raised limiting ridge in the stomach and one male treated with 30 mg/kg bw/day had a raised limiting ridge in the stomach, thickening of the non-glandular region of the stomach and sloughing in the stomach. No such effects were detected in females treated with 100 or 30 mg/kg bw/day.

Organ Weights

There were no toxicologically significant effects detected in the organ weights measured.

Histopathology

The following treatment related microscopic abnormalities were detected: Stomach: Hyperplasia, including hyperkeratosis, of the non-glandular stomach, of minimal to moderate severity, was noted in nine males and eight females treated with 600 mg/kg bw/day. Hyperkeratosis alone was present in two males and two females treated with 100 mg/kg bw/day. Ulceration of the non-glandular stomach was present in one male treated with 30 mg/kg bw/day. Erosion of the glandular mucosa (usually near the limiting ridge), of minimal severity, was noted in one male treated with 30 and 100 mg/kg bw/day and three males and four females treated with 600 mg/kg bw/day. Focal foveolar hyperplasia of the glandular mucosa, of minimal severity, was present in five females treated with 600 mg/kg bw/day. Focal inflammatory cell infiltrate in the glandular region, of minimal or mild severity, was noted in one male treated with 30 and 100 mg/kg bw/day and three males and five females treated with 600 mg/kg bw/day.

Conclusion

Based on the results of this study, the NOAEL was 600 mg/kg bw/day for both sexes.  There were some findings on the study were either sporadic and did not reflect an association with the dose of test substance, or were secondary effects caused by the irritant properties of the test substance.  

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion