Arabinofuranosidase, α-l-

Endpoint summary

Description of key information

The oral administration of arabinofuranosidase, batch PPH40331 to Han Wistar rats at doses up to 100% of the arabinofuranosidase batch (equivalent to 1116 mg TOS/kg/day) for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the arabinofuranosidase, batch PPH40331 (equivalent to 1116 mg TOS/kg/day).

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 that toxicologically relevant is unrealistic, due to the formulation of enzymes and the stringent work practices enforced because of the risk of sensitisation by inhalation.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-12-2015 to 07-07-2016

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)

Version / remarks:

revised 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

RccHanTM:WIST rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 41-47 days
- Weight at study initiation: 121 to 173 g for males; 113-148 g for females
- Fasting period before study: None
- Housing: 5 animals of the same sex per cage
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry).
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: 11 days

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

IN-LIFE DATES: From: 18 January 2016 To: 18 April 2016

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

reverse osmosis water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test enzyme was provided deep-frozen in a number of containers. The day before use, the required numbers of test material containers were transferred from the freezer to nominally 4ºC storage to allow them to thaw. On the day of formulation the test material containers were inverted 10 times and gently magnetically stirred. The required amount of test material was measured out and the residue returned to the freezer as quickly as practicable. The required volume of vehicle was added to the pre-measured material and gently mixed by magnetic stirring and inversion to avoid any potential damage to the protein and ensure homogeneity of the dose preparation. Formulations were retained refrigerated (2-8°C) overnight (when prepared the day before) or at ambient temperature.
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 112, 368 and 1116 mg total organic solids (TOS) /kg bw/day.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Reverse osmosis water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose samples were analysed according to GLP.
Stability of formulations at 10 and 100%, when retained for up to 24 hours at refrigerated (nominally 5°C) or ambient temperature was demonstrated b y Novozymes A/S. Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analysed for achieved concentration of the test substance. A total of 6 x 10 mL samples were taken from the middle of each formulation and all samples were frozen at approximately -20°C upon completion of sampling. Three samples from each formulation/occasion were subsequently dispatched (deep frozen on dry ice) to the Principal Investigator responsible for formulation analysis. All remaining samples were retained (at approximately -20°C) at Envigo as a contingency but were discarded after finalization of the formulation analysis report since no further analysis was required.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

112 mg/kg bw/day (actual dose received)

Remarks:

TOS

Dose / conc.:

368 mg/kg bw/day (actual dose received)

Remarks:

TOS

Dose / conc.:

1 116 mg/kg bw/day (actual dose received)

Remarks:

TOS

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The test enzyme was not anticipated to cause any significant findings, based on the results obtained from studies with similar materials. The highest dose (100%) is the maximum practical dose and represents administration of the enzyme, as received, at a volume dosage of 10 mL/kg body weight. The lower doses were selected using a ratio of approximately 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: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the treatment period.

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: All animals before treatment. Control and highest dose group after treatment.

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)
Platelet count (Plt)
Prothrombim time (PT)
Activated partial thromboplastin time (APTT)

Differential WBC count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)

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: The macroscopic examination of animals killed after 13 weeks did not reveal any findings that were attributable to treatment with Arabinofuranosidase, batch PPH44301. The nature and incidence of the findings were consistent with the commonly seen background of macroscopic changes in Han Wistar rats at these laboratories.
HISTOPATHOLOGY: Yes. The microscopic examination performed after 13 weeks of treatment revealed no changes related to treatment with Arabinofuranosidase, batch PPH40331 in treated animals. All histological findings were consistent with the normal histopathological findings in Han Wistar rats at these laboratories and were therefore considered incidental and unrelated to the test item.

Other examinations:

FAECAL ANALYSIS: No

Hematology, Bone Marrow: Yes

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy

Statistics:

The main tests used were Dunnett’s test, Shirley’s test, Williams’ test. Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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

Description (incidence and severity):

Males receiving 100% arabinofuranosidase, batch PPH40331, had a slightly higher incidence of faint superficial corneal opacities than controls but this was also apparent before treatment commenced and was therefore clearly not related to treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

A small number differences from controls attained statistical significance but these were either minor, confined to one sex and/or lacked dose-relationship and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

A number of inter-group differences from controls occurred, some of which attained statistical significance, but these were either minor, confined to one sex and/or lacked dose-relationship and were therefore attributed to normal biological variation.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

A number of inter-group differences from controls occurred, some of which attained statistical significance, but these were either minor, confined to one sex and/or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included increased adjusted kidney and liver weights in males given 100% arabinfuranosidase, batch PPH40331 where the increased were minimal, were not observed in females, and were therefore considered of no toxicological significance.

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:

>= 1 116 mg/kg bw/day (nominal)

Based on:

other: total organic solids (TOS)

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen so NOAEL was the highest dose administered.

Critical effects observed:

no

Conclusions:

It was concluded that oral administration of arabinofuranosidase, batch PPH40331 to Han Wistar rats at doses up to 100% of the arabinofuranosidase batch (equivalent to 1116 mg TOS/kg/day) for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the arabinofuranosidase, batch PPH40331 (equivalent to 1116 mg TOS/kg/day).

Executive summary:

The objective of this study was to assess the systemic toxic potential of arabinofuranosidase, batch PPH40331 (an enzyme used in the food industry), when administered orally by gavage to Han Wistar rats for 13 weeks. Three groups, each comprising 10 males and 10 females, received doses of 10, 33 or 100% of the arabinofuranosidase, batch PPH40331 (equivalent to 112, 368 or 1116 mg TOS/kg/day). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume-dose (10 mL/kg body weight).

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption (by daily visual observation), ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

General appearance and behaviour, sensory reactivity responses, grip strength and motor activity were not affected by treatment and there were no deaths during the study. There was no effect of treatment on body weight gain or on food and water consumption. There were no treatment-related ophthalmic findings. The haematology and blood chemistry investigations during Week 13 did not identify any toxicologically significant differences from controls. Organ weights were unaffected by treatment and there were no treatment-related macroscopic or histopathological findings.

Conclusion

It was concluded that oral administration of arabinofuranosidase, batch PPH40331 to Han Wistar rats at doses up to 100% of the arabinofuranosidase batch (equivalent to 1116 mg TOS/kg/day) for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the arabinofuranosidase, batch PPH40331.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 116 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:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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:

study scientifically not necessary / other information available

Justification for data waiving:

other:

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:

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:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The oral administration of arabinofuranosidase, batch PPH40331 to Han Wistar rats at doses up to 100% of the arabinofuranosidase batch (equivalent to 1116 mg TOS/kg/day) for 13 weeks was well-tolerated and did not cause any adverse change. The no-observed adverse-effect level (NOAEL) was considered to be 100% of the arabinofuranosidase, batch PPH40331 (equivalent to 1116 mg TOS/kg/day).

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 that toxicologically relevant is unrealistic, due to the formulation of enzymes and the stringent work practices, enforced because of the risk of sensitisation by inhalation.

Justification for classification or non-classification

Arabinofuranosidase is not classified.