Registration Dossier

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:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See any other information on materials and methods below
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Substance type: UVCB
- Name of test material (as cited in study report): Glucose Oxidase
- Lot/batch No.: R-GOx-04004
- Physical state: Clear brown liquid

Test animals

Species:
rat
Strain:
other: Ntac:SD
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Taconic M&B A/S, Ejby, Denmark
- Age: 4 to 5 weeks old at start of acclimation period
- Weight at study initiation: 191-192 g for males and 152-153 g for females
- Housing: The rats were kept in transparent polycarbonate cages (floor area: 810 cm²) with two in each cage, males and females separated. The cages were cleaned and the bedding changed at least twice per week.
- Diet: Pelleted rodent diet, ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3°C
- Humidity: 55 ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
1 mL stock solution contained 36.24 mg total protein. The test substance (as stock solution) was stored frozen at approximately -18°C until use. Before use, each bottle of stock solution was thawed to divide the contents into portions suitable for weekly preparation of dose formulations and frozen again. The stock solutions (original bottles or portions) were thawed overnight in the refrigerator. Before dividing the contents of the original bottles into portions and before preparation of the dose formulations, the stock solutions were stirred gently for at least 10 minutes on a magentic stirrer.

Group 1 (0 mg total protein/kg) was dosed with vehicle. The dose formulations for Groups 2, 3 and 4 (1.80, 3.60, and 10.87 mg total protein/kg, respectively) were prepared weekly by diluting the test substance (stock solution) with vehicle.

Dose formulations were prepared as follows:
Group 1: Vehicle
Group 2: 0.05 ml stock solution + 4.95 ml water for injection
Group 3: 0.10 ml stock solution + 4.90 ml water for injection
Group 4: 0.30 ml stock solution + 4.70 ml water for injection
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
In Weeks 1, 6 and 13, duplicate samples of the four dose formulations were taken into a 1.8 mL Cryotube and stored frozen at approximately -18°C and subsequently sent with dry ice to the sponsor, for analysis. The results are included in the raw data.
Duration of treatment / exposure:
91 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1.8 other: mg total protein/Kg
Remarks:
Concentration: 0.36 mg/mL
Dose / conc.:
3.6 other: mg total protein/Kg
Remarks:
Concentration: 0.72 mg/mL
Dose / conc.:
10.87 other: mg total protein/Kg
Remarks:
Concentration: 2.17 mg/mL
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- All visible signs of ill health and any behavioural changes were recorded daily during the morning hours. An additional morbidity/mortality check was performed in the afternoon.

DETAILED CLINICAL OBSERVATIONS
- Beginning prior to start of treatment, detailed clinical observations were performed outside the home cage once per week at similar times.

BODY WEIGHT
- All animals were weighed on arrival (Day -6), on the first day of treatment (Day 1) and weekly thereafter. Also the weight at necropsy was recorded.

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Weekly

OPHTHALMOSCOPIC EXAMINATION
- Before start of treatment, ophthalmoscopy was performed on all animals. Before termination of treatment (Week 12), all animals in Groups 1 and 4 were re-examined.

HAEMATOLOGY
- Time schedule for collection of blood: Before termination of treatment
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- Parameters checked in table No. 1 were examined.

CLINICAL CHEMISTRY
- Time schedule for collection of blood: Before termination of treatment
- Animals fasted: Yes
- Parameters checked in table No.2 were examined.

URINALYSIS
- Time schedule for collection of urine: Before termination of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.3 were examined.
Sacrifice and pathology:
GROSS PATHOLOGY: A macroscopic examination was performed after opening the cranial, thoracic and abdominal cavities and by observing the appearance of the organs and tissues in situ. Any macroscopic change was recorded with details of the location, colour, shape and size. Paired organs were weighed together. The relative organ weights were calculated for each animal. All tissues were fixed in phosphate buffered neutral 4% formaldehyde with the exception of the eyes (Davidsons's fixative) and testes (Bouin's fixative). The fixative for long term preservation was phosphate buffered neutral 4% formaldehyde for all tissues. The lungs were infused with fixative at necropsy.

HISTOPATHOLOGY: Parameters checked in table No.4 were examined. All organs from all control and high dose animals were examined microscopically. Submandibular lymph nodes with macroscopic visible signs of accumulation of blood due to blood sampling from the ipsilateral orbita venous plexus were fixed but not processed histologically. Both eyes were fixed but only the eye opposite the side used for blood sampling was examined microscopically. Tissues not examined microscopically were stored in fixative.
Other examinations:
Open field and stimuli-induced tests: During Weeks 12 and 13 of the study, all animals were examined with respect to reactivity to different types of stimuli (i.e. auditory, visual, tactile), grip strength and motor activity (open field test).
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Thereafter each continuous variable was tested for homogeneity of variance with Bartlett's test. If the variance was homogeneous, analysis of variance was carried out for the variable. If any significant differences were detected, possible inter-group differences were assessed with Dunnett's test (comparing treated groups with a control group). If the variance was heterogeneous, each variable was tested for normality by the Shapiro-Wilk method. In case of normal distribution, possible inter-group differences were identified with Student's t-test. Otherwise the possible inter-group differences were assessed by Kruskal-Wallis's test. If any significant inter-group differences were detected, the subsequent identification of the groups was carried out with Wilcoxon Rank-Sum test.
Ranked type of urine analysis data was analysed with Wilcoxon Rank-Sum test.
For all tests, the level of significance was defined as p <0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One high dose male was found dead on Day 29 before dosing. Necropsy findings indicated a probable mis-dosing as red lungs and fluid in the chest cavity was found.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
In Week 10, the males at 3.60 mg total protein/kg had statistically significantly lower food consumption when compared to the males of the control group. Since this finding was sporadic, without clear dose dependency and without a similar tendency in the opposite sex, it was considered to be incidental.
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Haematology revealed a statistically significant increase in red blood cell count (absolute) and statistically significant decrease in the mean cell haemoglobin and mean cell volume of males at 10.87 mg total protein/kg when compared to the males of the control group. In the absence of any pathology findings and similar findings in females, the biological significance of increased red blood cell count, decreased mean cell volume and decreased mean cell haemoglobin (noted in males at 10.87 mg total protein/kg) is uncertain. While statistical analysis of eosinophils revealed significant differences for males at 1.80 mg total protein/kg, this was restricted to a single sex and seen in the low dose level only, and therefore this is considered not to be attributable to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
At termination, a statistically significant decrease for cholesterol was seen in females at 1.80 mg total protein/kg compared to the control group. As this was seen in one sex only, and seen in the low dose level only, it is not considered to be attributable to treatment.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
At termination, a statistically significant decrease for potassium and chloride was seen in females at 1.80 mg total protein/kg compared to the control group. At the microscopic examination of the urine the incidence of bacteria was statistically significant higher forfemales at 1.80 mg total protein/kg compared to the control group. As these findings were seen in one sex and in the low dose level only, they are not considered to be related to treatment.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All findings reported were within the background of findings reported in this age and strain of laboratory maintained rats and as such considered to be of no toxicological significance. One males at 10.87 mg total protein/kg was found dead on Day 29 before dosing. Findings indicated a probable mis-dosing as red lungs and fluid in the chest cavity was found.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All findings reported were within the background of histological findings reported in this age and strain of laboratory maintained rats and as such considered to be of no toxicological significance. Pleural inflammation/subpleural alveolitis and intra alveolar haemorrhage/oedema was reported in the lungs of found dead male rat.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No effects observed in open field and stimuli-induced tests.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
10.87 other: mg total protein/Kg b.wt./Day
Sex:
male/female
Remarks on result:
other: (19.53 mg TOS/kg b.wt/Day)

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (Rat): 10.87 mg total protein/kg b.wt./Day (19.53 mg TOS/kg b.wt/Day)
Executive summary:

This study was conducted to assess the toxicity of the test substance administered daily by oral gavage to rats for 13 weeks. The study was conducted in accordance with the OECD Guideline 408. The rat was selected as the test model because of its suitability in this type of study. Oral treatment was chosen to comply with the intended route of exposure in humans. The doses were selected by the Sponsor.

Eighty SPF Sprague Dawley rats (40 males and 40 females) of the stock Ntac:SD were used in this study. The animals were allocated to four groups (10 males and 10 females each) and treated once daily by oral gavage for 91 days with water for injection (control, Group 1), 1.80 mg total protein/kg b.wt/day (Group 2), 3.60 mg total protein/kg b.wt/day (Group 3) or 10.87 mg total protein/kg b.wt/day (Group 4). 1 ml stock solution contained 36.24 mg total protein or 65.1 mg TOS. The dose volume was 5 ml/kg b.wt.

Clinical signs were recorded daily. Detailed clinical observations were performed once weekly. During Week 12 and 13 of the study, the animals were examined for sensory reactivity, grip strength and motor activity. Ophthalmoscopy was performed on all animals before start of treatment, and on the animals of Groups 1 and 4 during Week 12 of the study.

Body weight and food consumption were recorded weekly. Before termination of treatment, blood samples were taken for haematology and clinical chemistry, and urine was collected for urinalysis. The animals were killed and subjected to a macroscopic necropsy. Specified organs/tissues were weighed, fixed and prepared for a histopathological examination.

No treatment-related findings were recorded at the clinical and behavioural examinations, on food consumption, body weights or at the ophthalmoscopic examination.

No treatment-related findings were observed on the parameters for serum biochemistry, urinalysis and on organ weights.

In Group 4 animals (dosed 10.87 mg total protein/kg), increased values of red blood cell count, reduced mean cell volume and mean cell haemoglobin revealed statistically significant differences. However, in the absence of any pathology findings the biological significance of these findings is uncertain.

Necropsy and the following microscopic examination revealed no treatment-related effects.

In conclusion, statistical differences were seen in haematology as increased red blood cell count, decreased mean cell volume and decreased mean cell haemoglobin in males at 10.87 mg total protein/kg b.wt./Day. However, in the absence of any pathology findings and similar findings in females, the biological significance of this is uncertain.

The NOAEL (No observed adverse effect level) in this study was 10.87 mg total protein/kg b.wt./Day (19.53 mg TOS/kg b.wt/Day).