Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 December 2015 - 12 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to OECD test guideline no. 408 (1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is also used for production of food in EU.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
revised 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
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
- Physical state: liquid
- Lot/batch No.: PPQ40100
- Expiration date of the lot/batch: 03 November 2025
- Stability under test conditions: The test material and dilutions in water (10% and 100%) are stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Age at study initiation: 42-48 days
- Weight at study initiation: 141-184 g for males; 117-157 g for females
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-01-13 To: 2016-04-14

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 105, 347 and 1051 mg total organic solids (TOS) per kg body weight per day.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Water for formulation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP.
Analysis of achieved concentration with regard to % total nitrogen was performed on samples taken once during weeks 1, 6 and 13.
These results were similar to the expected concentrations for all three dose groups, demonstrating satisfactory formulation.

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
1 other: mL/kg/day
Remarks:
equivalent to 0.105 g TOS/kg/day
Dose / conc.:
3.3 other: mL/kg/day
Remarks:
equivalent to 0.347 g TOS/kg/day
Dose / conc.:
10 other: mL/kg/day
Remarks:
equivalent to 1.051 g TOS/kg/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected on the basis of results from studies performed on other similar enzyme preparations. The highest dose (10 mL/kg/day) was the maximum practical dose and represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg body weight, which is the maximum practical volume-dose for repeat dose oral administration. The lower doses were selected using an approximate ratio of 3.3 between doses.


Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Weekly group mean food consumptions and standard deviations were derived from unrounded cage values. Overall mean food consumption values were calculated from the weekly group mean values.

WATER CONSUMPTION:
- Time schedule for examinations: Daily by visual observation.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 12
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Prothrombim time (PT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : yes
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full macroscopic examination
HISTOPATHOLOGY: Yes. Adrenals, aorta, brain, intestines, epididymides, eosophagus, femur with joint, heart, kidneys, liver, lungs, selected lymphnodes, ovaries, pancreas, Peyer's patches, pituitary, prostate, salivary glands, skin with mammary gland, spinal cord, spleen, sternum, stomach, testes, thyroid, trachea, urinary bladder, uterus and vagina were preserved and for the control group and the high dose group these tissues were examined.
Other examinations:
FAECAL ANALYSIS: No

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy
Statistics:
Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, organ weights and any data derived from these was performed. The level of probability chosen as significant was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
One male rat from the low dose group was killed for welfare reasons in Week 8 after sustaining trauma to the tibio-tarsal joint of the right hind limp.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 051 mg/kg bw/day (nominal)
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) in rats was 10 mL of the undiluted xylanase/kg body weight/day equivalent to 1051 mg Total Organic Solids (TOS)/kg body weight/day.
Executive summary:

The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 408 (revised 1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is used for production of food in EU.

In conclusion, oral administration (by gavage) of xylanase to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted test material/kg body weight/day or 1051 mg Total Organic Solids (TOS)/kg body weight/day for thirteen weeks was well-tolerated and did not produce any toxicologically significant changes.

Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL xylanase/kg body weight/day or 1051 mg Total Organic Solids (TOS)/kg body weight/day.