Cellulase

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 28, 2011 – January 6, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD guidelines 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme protein of Cellulase (EC no. 232-734-4, CAS no. 9012-54-8, EC name: Cellulase, Enzyme Class no. 3.2.1.4)

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

solid

Details on test material:

Substance type: UVCB
Physical state: solid
Stability under test conditions: Stability of Test Item in Vehicle: 1 day at room temperature. Stability of Dose Formulations: 8 days in the refrigerator based on results obtained in this study.
Storage conditions of test material: At room temperature (20 ± 5 °C)
Storage of Dose Formulations during Application: At room temperature (20 ± 5 °C) in glass beakers.
Storage of Dose Formulations (when prepared weekly): In the refrigerator (5 ± 3 °C) in glass beakers.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst /Netherlands
Number of Animals:
Group 1: 10 males and 10 females
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 10 males and 10 females
Group 10: 2 males and 2 females*
Total Number of Animals Used: 40 males and 40 females
Total Number of Animals Ordered: 42 males and 42 females
Age (at Delivery): 7 weeks
Body Weight Range (at Acclimatization):
Males: 168.1 g to 183.6 g
Females: 140.1 g to 162.5 g
Identification: Acclimatization: Cage card and tail mark (later ear tattoo)
Identification: Treatment: Cage card and individual ear tattoo
Randomization: Randomly allocated to groups by body weight.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

* Reserve animals were exchanged as needed and then removed from the study.


Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, and are considered not to have any influence on the study. The data are retained at Harlan Laboratories Ltd. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

bidistilled

Details on oral exposure:

Test material (B-Glucanase produced with Trichoderma reesei) was administered daily by oral gavage in bidistilled water vehicle

Dose levels were 100, 300 and 1000 TOS mg/kg body weight/day for a period of 13 weeks (92/93 days)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The application formulations investigated during the study were found to comprise ß-Glucanase in the range of 81.2% to 99.0%, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of ß-Glucanase produced within the preparations was approved because single results did not deviate more than 5.8% (<15%) from the corresponding mean.

In addition, the test material was found to be stable with reference to the TOS method in application formulations when kept five hours at room temperature or eight days in the refrigerator due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean.

In conclusion, the results indicate the accurate use of the test item ß-Glucanase and purified water as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.


Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily
Daily Dose Levels in Total Organic Substance (TOS):
Group 1: 0 mg/kg body weight/day
Group 2: 100 mg/kg body weight/day
Group 3: 300 mg/kg body weight/day
Group 4: 1000 mg/kg body weight/day
Rationale for Dose Level Selection:The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats,
Harlan Laboratories study D29560 (non-GLP).
Dose Volume: 10 mL/kg body weight
Dose Concentrations (TOS):
Group 1: 0 mg TOS/mL
Group 2: 10 mg TOS/mL
Group 3: 30 mg TOS/mL
Group 4: 100 mg TOS/mL
Dose Concentrations (actual):
Group 1: 0 mg/mL
Group 2: 11.02 mg/mL
Group 3: 33.06 mg/mL
Group 4: 110.2 mg/mL

Duration of Acclimatization Period: 6 days
Duration of Treatment Period: 13 weeks

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 TOS mg/kg body weight/day
Basis:
nominal in water

No. of animals per sex per dose:

10 female and 10 male rats

Control animals:

yes, concurrent vehicle

Details on study design:

Test item B-Glucanase produced with Trichoderma reesei was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300, and 1000 mg/kg body weight/day for a period of 13 weeks (92/93 days). A control group was treated similarly with the vehicle, bidistilled water, only. Each group was sacrificed after 92 or 93 days of treatment.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 and weekly thereafter, detailed observations were recorded

BODY WEIGHT: Yes
Time schedule for examinations: weekly

FOOD CONSUMPTION:
Food consumption for each cage, i.e. sum of five animals, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes

FOOD EFFICIENCY:
Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
Time schedule for examinations: weekly, over a 3-day period in each week

OPHTHALMOSCOPIC EXAMINATION: Yes
Time schedule for examinations: Before treatment started and during week 13
Dose groups that were examined: control and highest dose group

CLINICAL CHEMISTRY: Yes
No test item-related clinical signs were observed in all groups.

HEMATOLOGY: Yes
At the end of the treatment period (week 13), blood samples were withdrawn for hematology and plasma biochemistry analyses. The test animals were placed in metabolism cages for 18 hours prior to sample collection.

URINALYSIS: Yes
At the end of the treatment period (week 13), urine samples were collected for urinalysis. Urine was collected during an 18-hour period that the animals were placed in metabolism cages.

NEUROBEHAVIOURAL EXAMINATION: Yes
At the end of the treatment period (week 13), high dose and control groups were examined for sensory activity, grip strength, and motor activity

Sacrifice and pathology:

At the end of the treatment period (week 13 on day 92 or 93), fasting blood samples were withdrawn for hematology and plasma biochemistry analyses. Urine samples were collected for urinalysis. The test animals were placed in metabolism cages for 18 hours prior to blood sampling. Urine was collected for analysis during this time period. All animals were then sacrificed, necropsied and examined post mortem. Isoflurane anesthesia was used in sacrificing the test subjects. Gross pathology and histopathology was performed. Histological examinations were performed on organs and tissues from all control and high dose animals.

Other examinations:

FAECAL ANALYSIS: No

Weight of individual organs: yes
Time schedule for collection of organs: At necropsy

Statistics:

The following statistical methods were used to analyze grip strength, locomotor activity, food consumption, body weight, ophthalmoscopic examinations, clinical laboratory data, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs were observed during the daily cage side observations as well as the weekly detailed behavioral observations in all groups.

Mortality:

no mortality observed

Description (incidence):

No clinical signs were observed during the daily cage side observations as well as the weekly detailed behavioral observations in all groups.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

All gross lesions recorded were considered to be within the range of normal background alterations.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The changes that were identified were not considered of toxicological significance.

Histopathological findings: neoplastic:

not specified

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 013.3 mg/kg bw/day (nominal)

Based on:

other: Enzyme concentrate dry matter

Basis for effect level:

other:

Remarks on result:

other: NOAEL and NOEL is set at the highest dose.

2

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

other: Total organic solids (TOS)

Basis for effect level:

other: There were no adverse effects observed at the highest dose tested.

Remarks on result:

other: NOAEL and NOEL is set at the highest dose.

3

Dose descriptor:

NOEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

other: Total organic solids (TOS)

Basis for effect level:

other: There were no adverse effects observed at the highest dose tested.

Remarks on result:

other: NOAEL and NOEL is set at the highest dose.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

One control male (no. 6) died spontaneously on day 45 of treatment. Before death, decreased activity, ruffled fur and prostrate appearance were noted. The cause of death could not be established. All other animals survived the scheduled treatment period.

 

Clinical Signs (Daily Cageside Observations): No test item-related clinical signs were observed in all groups.

 

Detailed Behavioral Observations (Weekly): No findings were recorded at the detailed behavioral observations during acclimatization and treatment weeks 1 to 12.

 

Functional Observational Battery: No clinical signs were evident between the high dose group and the control group during the functional observational battery at week 13.

 

Grip Strength: No test item-related changes in grip strength were noted. Significantly increased grip strength was recorded on the fore limb in females at 300 and 1000 mg/kg/day and decreased grip strength on the hind limb in females and males at 1000 mg/kg/day. As a contrary effect on fore and hind limbs was observed and no clinical sings or changes in locomotor activity were recorded, this was considered to be incidental.

Locomotor Activity: No test item-related differences in locomotor activity were noted between the groups. Significant differences compared to control were noted on individual measurements in males and females of the low and mid dose groups and showed no dose dependency. Therefore, these differences were considered not to be test item-related.

 

Food Consumption: There were no test item-related effects on food consumption.

 

Body Weights: No differences in body weight development were noted between the groups.

 

Ophthalmoscopic Examinations: There were no test item-related ophthalmoscopic findings.

Hematology: No test item-related hematological findings were noted. Collection was performed at week 13 from all study animals after fasting.

Urinalysis: No effects on urine parameters were noted at any dose level. Collection was performed at week 13 from all study animals after fasting.

Clinical biochemistry: No test item-related changes in clinical biochemistry were noted. Collection was performed at week 13 from all study animals after fasting.

Organ weights: No test item-related changes in the mean absolute and relative organ weights were recorded.

Macroscopic findings: All gross lesions recorded were considered to be within the range of normal background alterations.

Microscopic findings:Under the conditions of this study, the test item did not show any morphological evidence of toxicological properties in the organs and tissues examined.

Applicant's summary and conclusion

Conclusions:

Oral administration (gavage) of ß Glucanase to Wistar rats at doses of 100, 300 and 1000 mg/kg/day for a period of 13 weeks was generally well tolerated.

No test item-related deaths occurred and no changes in daily or weekly observations as well as functional observational battery (performed during week 13) including grip strength and locomotor activity were observed. Furthermore, no test item-related effects on body weights or food consumption, no ophthalmoscopic findings, no changes in clinical laboratory investigations of toxicological relevance as well as no macroscopic and microscopic findings were noted.

Based on the results of this study, the no observed effect level (NOEL) as well as the no observed adverse effect level (NOAEL) for the test item ß-Glucanase was set at 1000 mg/kg/day TOS which is equivalent to 1013.3 mg enzyme concentrate dry matter/kg bw/day = 469 mg active enzyme protein/kg bw/day.

Executive summary:

The repeated dose oral toxicity study was a 13-week subchronic toxicity study conducted in rats according to OECD guideline No. 408 (revised in 1998) and in compliance with GLP.

The test material used in this study, β-glucanase (batch No. CE 10088B3) is dissolved in bi-distilled water to yield concentration of 10, 30 and 100 mg TOS/ml. The test material was given to 10 animals/sex/group in a constant volume of 10 ml/kg bw by gavage to achieve the desired concentrations of 100, 300 and 1000 mg TOS/kg bw/day for 90 consecutive days. Bi-distilled water given at the same volume served as vehicle control. The test material in solution was tested for stability, homogeneity and dosing verification throughout the study. Clinical observations were made daily whereas feed, water and body weight were recorded weekly. Ophthalmologic examinations were done prior to study initiation and at study termination. Clinical chemistry, hematology, urinalysis, FOB (Functional Observational battery), necropsy, organ weights, and histopathologic examinations were conducted at study termination as required by the guideline.

One control animal died on day 45 but this death was considered as incidental and non-treatment related. There were no treatment related effects found in all parameters investigated. In summary, oral administration of β-glucanase (cellulase) to rats of both sexes at dose levels of 100, 300 and 1000 mg/kg bw/day (expressed as Total Organic Solids [TOS]) for 13 consecutive weeks did not result in any systemic, behavioral or pathological changes. The No Observed Adverse Effect Level (NOAEL) was therefore established at the highest dose tested, 1000 mg β-glucanase (cellulase)/kg body weight/day (expressed as TOS) which is equivalent to 1013.3 mg enzyme concentrate dry matter/kg bw/day = 469 mg active enzyme protein/kg bw/day.