1,4-α-glucan branching enzyme

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2007 to 13 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of 1,4-alpha-glucan branching enzyme (CAS no.9001-97-2 , EC name 1,4-alpha-glucan branching enzyme, Enzyme class no. 2.4.1.18)

Molecular formula:

n.a.

IUPAC Name:

Active enzyme protein of 1,4-alpha-glucan branching enzyme (CAS no.9001-97-2 , EC name 1,4-alpha-glucan branching enzyme, Enzyme class no. 2.4.1.18)

Constituent 2

Reference substance name:

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

Molecular formula:

Not available

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:

liquid

Details on test material:

- Lot/batch No.: PPY27209
- Expiration date of the lot/batch: June 2017

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: approximately 6 week
- Weight at study initiation: Weighed 135.2 g (mean for females) and 179.6 g (mean for males)
- Fasting period before study: None
- Housing: In Macrolon cages (480 x 375 x 210 mm) with sterilized wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment.
- Diet: Rat & Mouse No. 3 Breeding Diet, RM3; pelleted
- Water: Ad libitum. Water, taken from the public supply.
- Acclimation period: 13 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

and purified water for the other control

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials (one vial per group per day) in a freezer at < -18ºC.
VEHICLE
- The vehicle for dosing the controls was stored as described above.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared for administration on one occasion in Week 1 and 13 were taken for content check analysis . The results of these analyses indicated values close and acceptable to the intended enzyme activity.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

113 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Dry Matter

Doses / concentrations 2

Dose / conc.:

372 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Dry matter

Doses / concentrations 3

Dose / conc.:

1 128 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Dry matter

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: 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.

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual observations of all animals were also recorded before and after dosing on each day of treatment as indicated below. The timing of these observations were performed to establish and confirm any pattern of signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded during the acclimatisation period, on the day that treatment commenced, at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of the amount spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Were made prior to the start of treatment in all rats, and in the last week of the treatment period in all rats of the control group and the high-dose group.
- Dose groups that were examined: Observations were bilateral unless otherwise indicated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
Differential leucocyte counts were determined automatically by counting the numbers of lymphocytes, neutrophils, monocytes, eosinophils, basophils and large unstained cells in the instrument sample.

CLINICAL CHEMISTRY: Yes
The concentration of each protein fraction was determined by reference to the percentage value and to the total protein concentration. Albumin to globulin (A/G) ratios were calculated from the percentage values before conversion to absolute concentrations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes

Statistics:

The main tests used were analysis of covariance (ANOVA), Shapiro-Wilks test, Dunnett's multiple comparison test, Kruskal-Wallis non-parametric analysisi, Pearson chisquare analysis, and Fisher's Exact Probability test. Significant differences between the groups compared were expressed at the 5% (p<0.05) level.

Results and discussion

Results of examinations

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

Description (incidence and severity):

The amount of food consumed by males receiving 0.5 g TOS/kg/day was slightly lower than that of the purified water control group but, in the absence of a similiar finding among those receiving the highest dosage, this was considered fortuitous.

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

Description (incidence and severity):

Only a significantly lower mean corpuscular haemoglobin concentration (MCHC) in high-dose males were observed. This change was not seen in females and was not estimated to be test item related.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

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):

The following statistically significant differences in organ weights were observed compared to controls: 1) Absolute brain weight was significantly lower in males of the high-dose group, 2) Relative liver weight was significantly higher in males of the high-dose group and 3) Absolute and relative weight of the epididymides was significantly lower in the mid-dose group. None of these observations were considered test item related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Reactive hyperplasia of lymphocytic tissue in the mandibular lymph nodes appeared to be slightly increased in incidence and severity in the treated males, compared with the control groups, but the finding did not show a clear dose relationship.
Examination of the hearts of control and high dose males suggested that there may have been a treatment-related increased incidence of chronic myocarditis. However, when the examination was extended to the other controls and treated groups, the incidence seen at 2.6 g TOS/kg/day was similar to that in the control group. It was therefore concluded that treatment did not cause any change in the heart.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 128 mg/kg bw/day (nominal)

Based on:

other: Dry matter

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Undiluted concentrate of 1,4-alpha-glucan branching enzyme, batch PPY 27209 did not cause any adverse changes in any of the investigated parameters in the sub-chronic toxicity study in rats. Therefore, the no-observed-adverse-effect level (NOAEL) for 1,4-alpha-glucan branching enzyme, batch PPY 27209 was considered to be 1128 mg Dry matter/kg bw/day for both sexes.

Executive summary:

The objective of this study was to provide data on the possible sub-chronic toxicity of 1,4-alpha-glucan branching enzyme, batch PPY 27209 in rats of both sexes upon daily oral administration by gastric gavage for 13 consecutive weeks.

Clinical observations, neurobehavioural testing, growth, food and water consumption, ophthalmoscopy, haematological and clinical chemistry parameters, gross examination at necropsy, organ weights and microscopic examination of various organs and tissues were used as criteria for disclosing possible harmful effects.

No indication of toxicity of the test substance was observed after oral adminitration in rats, since none of the parameters investigated showed any treatment-related or toxicologically relevant changes.

In conclusion, undiluted concentrate of 1,4-alpha-glucan branching enzyme, batch PPY 27209 did not cause any adverse changes in any of the investigated parameters in the sub-chronic toxicity study in rats. Therefore, the no-observed-adverse-effect level (NOAEL) for 1,4-alpha-glucan branching enzyme, batch PPY 27209 was considered to be 1128 mg Dry matter/kg bw/day for both sexes.