Glucanase, endo-1,3(4)-β-

Endpoint summary

Description of key information

It was concluded that oral administration of beta-glucanase batch PPB24534, to Sprague Dawley rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicologically significant changes. Consequently, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 10.0 mL/kg/day, equivalent to 1266 mg TOS/kg/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:

05-01-2005 to 06-02-2006

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:

Adopted on 21 September 1998.

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Details on species / strain selection:

Ntac:SD strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic M&B A/S
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 6 weeks old
- Fasting period before study: None
- Housing: 2 animals of the same sex per cage
- Diet: A complete pelleted rodent diet "Altromin 1314" (for growing animals) was available ad libitum.
- Water: The animals had free access to bottles with domestic quality drinking water acidified with hydrochloric acid to pH 2.5 in order to prevent microbial growth.
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21°C ± 3°C
- Humidity: 55% ± 15% RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: I 5 February 2005 To: 18 May 2005

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

tap water (analysed for relevant possible contaminants)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared daily by diluting the test article in tap water. Treatment was completed within four hours after preparation of the dose formulations. The test article was kept frozen at approximately -18°C until use. Before use, each bottle of the test article was thawed to divide the contents into portions suitable for daily preparation of dose formulations and frozen again. The test article (original bottles or portions) was thawed overnight in the refrigerator or at room temperature for max. 4 hours before dosing the animals. Before dividing the contents of original bottles into portions and before preparation of the dose formulations, the test article was stirred gently for at least 10 minutes on a magnetic stirrer.
VEHICLE
- Concentration in vehicle:
Group 1: Vehicle (tap water).
Group 2: 1 part test article diluted in 9 parts vehicle.
Group 3: 1 part test article diluted in 2.03 parts vehicle.
Group 4: Undiluted test article.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Tap water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In Weeks 1, 6 and 13, triplicate (3) samples of the four dose formulations were taken and stored frozen at approximately -18°C and subsequently sent to Novozymes A/S for analysis.
Dose samples were analysed according to GLP.
36 samples were received at Novozymes A/S frozen. The samples were stored frozen -18°C and thawed at room temperature at the day of the analysis.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

Given as total organic substance (TOS)

Dose / conc.:

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

Remarks:

Given as total organic substance (TOS)

Dose / conc.:

1 266 mg/kg bw/day (actual dose received)

Remarks:

Given as total organic substance (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: Daily during morning hours.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: An additional morbidity/mortality check was performed in the afternoon. Beginning prior to start of treatment, detailed clinical observations were performed outside the home cage once per week at similar times.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals were weighed on arrival, on the day of re-allocation (Day 1) and weekly thereafter. Also the weight at necropsy was recorded.

FOOD CONSUMPTION:
- Starting Day 1, the consumption of food was recorded weekly for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups: Before start of treatment, ophthalmoscopy was performed on all animals. Before termination of treatment (Week 12), all animals in Groups 1 and 4 were re-examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- How many animals: From all animals
- Parameters checked:
Haemoglobin (Hb)
Red blood cell count (RBC)
Haematocrit (HT)
Mean cell volume (MCV)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
White blood cell count (WBC)
Differential leucocyte count (NEUTRO, LYMPHO, EOS, BASO, MONO)
Platelet count (Pit)
Activated partial thromboplastin time (APTT)
Prothrombin time (Pt)
Fibrinogen (Fib)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- How many animals: From all animals
- Parameters checked:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline phosphatase (ALKPH)
Bilirubin (total) (BILI)
Gamma-glutamyl transferase (GGT)
Cholesterol (CHOL)
Triglycerides (TRIG)
Carbamide (UREA)
Creatinine (CREAT)
Glucose (GLUC)
Sodium (Na)
Potassium (K)
Calcium (Ca)
Magnesium (Mg)
Inorganic phosphorus (P)
Chloride (Cl)
Protein (total) (PROTEIN)
Albumin (ALB)
Globulin
Albumin/Globulin (ALB/G) ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Week 13
- Metabolism cages used for collection of urine: Yes
- Parameters checked:
Volume
Sodium (Na)
Potassium (K)
Chloride (Cl)
Specific gravity (SG)
pH
Colour (COLOUR)
Protein (PROTEIN)
Leucocytes (LEUC)
Blood (BLOOD)
Glucose (GLUCOSE)
Ketones (KETONES)
Bilirubin (BILI)
Urobilinogen (UROBIL)

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: On one occasion in 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:

Hematology, Bone Marrow: At necropsy, a bone marrow smear was taken from the femur of all animals The smears were fixed and stained with May-Grunwald and Giemsa stain. As the haematological findings did not indicate a need to do so, the smears were discarded.

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

Statistics:

The main tests used were Barlett's test, Dunnett’s test, Kruskal-Wallis test, Shapiro-Wilk method, Wilcoxon Rank-Sum test.
For all tests, the level of significance was defined as p<0.05. The statistical analyses were made with SAS® procedures (version 8.2).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The total body weight gain for Group 3 males was statistically significantly increased compared to the control group. For Group 3 females the body weight gain on Day 36 and Day 43 decreased statistically significantly compared to the control group. As this was seen at the intermediate dose level only and in both cases with no clear dose dependency, this is not considered to be related to treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

In Week 6, the males of Group 4 and the females of Group 3 had statistically significantly lower food consumption when compared to the control group. Since these findings were sporadic and without clear dose dependency, they were considered to be incidental.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

At termination, a statistically significant increase in cholesterol was seen in Group 3 males compared to the control group. As this was seen in one sex only, at the intermediate dose level and with no clear dose dependency this is considered to be without any toxicological significance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Treatment related effect was seen in the urine as statistically significantly decreased values for pH in both Group 4 males and females compared to the control group.
The increased incidences of lower pH and higher amount of urates in the urine of some of the female rats were considered to be related to treatment. The homeostatic role played by the kidney in water balance is well known and low urinary pH is known to be the most significant element in the generation of urine sediments. However, since the changes in urinalysis were within the range of values seen on other studies with similar compounds at these laboratories and because no kidney-related findings were observed in this study, the changes were considered to be of no toxicological importance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

At termination, in the males, the relative brain (Group 3) and kidney (Group 2) weight were statistically significant decreased compared to the control group. As this was seen in one sex and in the low and intermediate dose levels only this is not considered to be treatment related.

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:

>= 1 266 mg/kg bw/day (nominal)

Based on:

other: total organic solids (TOS)

Sex:

male/female

Basis for effect level:

urinalysis

Critical effects observed:

no

Conclusions:

It was concluded that oral administration of B-glucanase batch PPB24534, to Sprague Dawley rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicologically significant changes. Consequently, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 10.0 mL/kg/day, equivalent to 1266 mg TOS/kg/day.

Executive summary:

The objective of this study was to assess the toxicity of beta-glucanase, batch PPB24534 following daily oral gavage to rats for 13 weeks. The study was conducted in accordance with the OECD Guideline 408, adopted on 21 September 1998. The rat was selected as the test model because of its suitability in this type of study. Oral treatment was chosen to comply with the intended route of exposure in humans. The doses were selected by the Sponsor.

Eighty SPF Sprague Dawley rats (40 males and 40 females) of the stock Ntac:SD were used in this study. The animals were allocated to four groups (10 males and 10 females each) and treated once daily by gavage for at least 91 days with tap water (control, Group 1), 10% B-glucanase (Group 2), 33% B-glucanase (Group 3) or 100% B-glucanase (Group 4). This corresponded to 0, 126.6, 417.8 and 1266 mg total organic solids (TOS)/kg bw, respectively. The dose volume was 10 mL/kg bw. Clinical signs were recorded daily. Detailed clinical observations were performed once weekly. During Week 12 of the study, the animals were examined for sensory reactivity, grip strength and motor activity. Ophthalmoscopy was performed on all animals before start of treatment, and on the animals of Groups 1 and 4 during Week 12 of the study. Body weight and food consumption were recorded weekly. Before termination of treatment, blood samples were taken for haematology and clinical chemistry, and urine was collected for urinalysis. The animals were killed and subjected to a macroscopic necropsy. Specified organs/tissues were weighed, fixed and prepared for a histopathological examination.

No treatment related findings were recorded at the clinical and behavioural examinations, on food consumption, body weights or at the ophthalmoscopic examination. No treatment related findings were observed on the parameters for serum biochemistry, haematology and on organ weights.

Treatment related effect was seen in the urine as decreased values for pH in both Group 4 males and females and higher incidence of urates in Group 3 and 4 females compared to the control group. The toxicological significance of this observation is questionable, as the homeostatic role played by the kidney in water balance is well known and also because low urinary pH is known to be the most significant element in the generation of urine sediments. In the absence of any other kidney-related pathology findings, the changes were considered to be of no toxicological importance. Necropsy and the following microscopic examination revealed no treatment related effects.

It was concluded that oral administration of beta-glucanase, batch PPB24534, to Sprague Dawley rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicologically significant changes. Consequently, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 10.0 mL/kg/day, equivalent to 1266 mg TOS/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 266 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

It was concluded that oral administration of beta-glucanase batch PPB24534, to Sprague Dawley rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicologically significant changes. Consequently, the no-observed-adverse-effect level (NOAEL) in this study was considered to be 10.0 mL/kg/day, equivalent to 1266 mg TOS/kg/day.

Justification for classification or non-classification

Not classified.