Mannanase, endo-1,4-β-

Endpoint summary

Description of key information

The repeated dose oral toxicity of Mannanase has been tested by oral gavage in Han Wistar rats. Dosing of the animals at doses up to 100% of the Mannanase, batch PPE42634 (equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day) for 13 weeks was well-tolerated and there was no significant finding of any toxicological relevance. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 100% Mannanase, batch PPE42634 equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day.

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:

20 December 2016 to 13 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This assessment of sub-chronic systemic toxicity (according to OECD TG 408) has been performed due to data requirements from the European Food Safety Authority (EFSA), as this enzyme also comply with the regulatory system of FIAP [REGULATION (EC) No 1331/2008 and EFSA CEF guidance from 2009/2013]. Moreover; it has been generated in accordance with Directive 2010/63/EU.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

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: 160 to 200 g for males; 122-161 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: 15 days

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

IN-LIFE DATES: From: 21 December 2016 To: 26 May 2017

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, which were further divided (for highest group formulations) or used for dilution procedures to provide sufficient aliquots for each day of the study. Bulk containers were defrosted in a
refrigerator overnight prior to preparation of the daily aliquots for study use. The final study aliquots and any remaining test-item were returned to the freezer as soon as practical. Bulk containers were stirred using a magnetic stirrer gently to ensure homogeneity prior to preparation of the daily aliquots. Daily aliquots were prepared on a weekly basis. Daily aliquots were either stored at ambient temperature (in cases where the formulations were prepared and administered on the same day) or were stored frozen (-10 to -30°C) and either thawed overnight in a refrigerator (2 to 8°C), if dose administration was conducted on the following day, or thawed at ambient temperature if dose administration was conducted on the same day. In either case, formulations were allowed to equilibrate at room temperature before dosing and were administered within 24 hours of the commencement of thawing procedures.

- Concentration in vehicle: 10, 33 and 100%, corresponding to 128, 425 and 1277 mg enzyme concentrate dry matter/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 10 and 100% formulations when retained for up to 24 hours refrigerated (2 to 8°C) or at ambient temperature (15 to 25°C), and up to 168 hours (seven days) when stored deep-frozen (-10 to -30°C) were demonstrated by Novozymes A/S. Samples of each formulation prepared for administration in Weeks 1, 6 and 13 of treatment were analyzed for total nitrogen content (%).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

128 mg/kg bw/day (nominal)

Remarks:

mg enzyme concentrate dry matter/kg bw/day

Dose / conc.:

425 mg/kg bw/day (nominal)

Remarks:

mg enzyme concentrate dry matter/kg bw/day

Dose / conc.:

1 277 mg/kg bw/day (nominal)

Remarks:

mg enzyme concentrate dry matter/kg bw/day

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:

N/A

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 (Day 1), 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 study.

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: All aniumals before treatment started and during week 13 (animals from Groups 1 and 4).
- 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)
Hemoglobin (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total white cell count (WBC)
Platelet count (Plt)

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)
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

HISTOPATHOLOGY: Yes

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, Steel's test, Fisher's exact test and Barlett's 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

Haematological findings:

no effects observed

Description (incidence and severity):

All differences from controls, including those that attained statistical significance, were minor, confined to one sex, lacked dose-relationship and were within the background range and, consequently, were attributed to normal biological variation.
In males there was a single high dose male with a total leucocyte count that was slightly below the concurrent control values and just below the background range.
The statistically significantly reduced prothrombin times in females receiving 33% Mannanase was clearly incidental in the absence of a similar finding at the highest doseand with the majority (9/10) of values within the background range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The biochemical analysis of the plasma in Week 13 did not identify any difference from controls that was attributable to treatment. All differences from controls, including those that attained statistical significance, were minor, confined to one sex, lacked dose-relationship and were within the background range and, consequently, were attributed to normal biological variation.
At all doses there was a statistically significantly high plasma creatinine concentration in males but this was attributed to five control animals having values that were below the background range leading to a lower than expected group mean value for the control males.

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

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:

>= 1 277 mg/kg bw/day (nominal)

Based on:

other: mg enzyme concentrate dry matter

Sex:

male/female

Basis for effect level:

other: there are no adverse effects observed at the highest dose tested

Key result

Critical effects observed:

no

Conclusions:

It is concluded that oral administration of Mannanase, batch PPE42634 to Han Wistar rats at doses up to 100% of the Mannanase, batch PPE42634 (equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day) for 13 weeks was well-tolerated and there was no significant finding of any toxicological relevance. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 100% Mannanase, batch PPE42634 equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Mannanase, batch PPE42634 (an enzyme used in the food industry), when administered orally by gavage to Han Wistar rats for 13 weeks. Three groups, each comprising ten males and ten females, received doses of 10, 33 or 100% of Mannanase, batch PPE42634 (equivalent to 128, 425 and 1277 mg enzyme concentrate dry matter/kg body weight/day). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume-dose (10 mL/kg body weight) as the treated groups. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmic examination, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

There was no death and no clinical sign, behavioral finding that were attributable to treatment. In addition there was no effect of treatment on bodyweight or food and water consumption. There was no treatment-related ophthalmoscopic finding. The haematological analysis of the peripheral blood and biochemical analysis of the plasma in Week 13 did not identify any difference from controls that was attributable to treatment. Organ weights were unaffected after 13 weeks and there was no treatment related.

It is concluded that oral administration of Mannanase, batch PPE42634 to Han Wistar rats at doses up to 100% of the Mannanase, batch PPE42634 (equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day) for 13 weeks was well-tolerated and there was no significant finding of any toxicological relevance. The no-observed-adverse-effect level (NOAEL) in this study was considered to be 100% Mannanase, batch PPE42634 equivalent to 1277 mg enzyme concentrate dry matter/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 277 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 have been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints, 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 thus be considered of high quality.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

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 have been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints, 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 thus be considered of high quality.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A repeated dose oral toxicity study was conducted according to OECD guideline 408 (adopted 1998) and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, equivalent to 10 mL of undiluted test material/kg bw/day or 1277 mg enzyme concentrate dry matter/kg bw/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 and the large size 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. In addtion, the acute inhalation exposure of up to 450 mg enzyme concentrate dry matter/m3 did not show any adverse effects.

Justification for classification or non-classification

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