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Description of key information

The repeated dose oral toxicity of xylanase has been tested. The repeated dose inhalation and dermal toxicity were waived based on exposure considerations and the properties of the substance. The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested - 1051 mg/kg bw/day (based on TOS (Total Organic Solids)).

Based on repeated dose oral study and weight of evidence, xylanase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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Reference
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.
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
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
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
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
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.
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
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'
Key result
Critical effects observed:
no
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 051 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated dose oral toxicity of xylanase was conducted according to OECD guideline 408, in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, 1051 mg/kg bw/day (based on TOS (Total Organic Solids)).

The repeated dose inhalation and dermal toxicity studies were waived.

- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitization by inhalation.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: The production strains of xylanase meet the criteria for safe strain production micro-organisms. The conclusion of the no adverse effect level is supported by read-across from oral repeated dose toxicity of other glycosidase enzymes of IUBMB subclass 3.2.1, where xylanase also belongs. 

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint: The inhalation study was waived because exposure is too low to exert any toxicity. Strict exposure controls are based on the more sensitive health effect of respiratory sensitization. Potential exposure by inhalation to an amount of enzyme that is toxicologically relevant is unrealistic due to the stringent work practices and the formulation of enzymes.

 

Justification for selection of repeated dose toxicity inhalation - local effects endpoint: The inhalation study was waived because exposure is too low to exert any toxicity. Strict exposure controls are based on the more sensitive health effect of respiratory sensitization. Potential exposure by inhalation to an amount of enzyme that is toxicologically relevant is unrealistic due to the stringent work practices and the formulation of enzymes. 

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint: Skin irritation was not seen in any of the test animals in the single-dose study in rabbits. No signs of toxicity were noted. Repeated dosing was not performed and is unlikely to produce any systemic effects given the structure and properties of the enzyme protein. The overall conclusion that xylanase is non-toxic upon repeated dermal exposure is supported by the knowledge of low possibility of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein. 

Justification for selection of repeated dose toxicity dermal - local effects endpoint: Skin irritation was not seen in any of the test animals in the single-dose study in rabbits. No signs of toxicity were noted. Repeated dosing was not performed and is unlikely to produce any systemic effects given the structure and properties of the enzyme protein. The overall conclusion that xylanase is non-toxic upon repeated dermal exposure is supported by the knowledge of low possibility of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

Justification for classification or non-classification

Based on repeated dose oral study and weight of evidence, xylanase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.