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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 February 1995 to 14 June 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

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

Constituent 1
Reference substance name:
Active enzyme protein of Cyclomaltodextrin glucanotransferase (EC no. 618-522-8, CAS no.9030-09-5, EC name Cyclomaltodextrin glucanotransferase , Enzyme class no 2.4.1.19 )
Molecular formula:
n.a.
IUPAC Name:
Active enzyme protein of Cyclomaltodextrin glucanotransferase (EC no. 618-522-8, CAS no.9030-09-5, EC name Cyclomaltodextrin glucanotransferase , Enzyme class no 2.4.1.19 )
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:
liquid
Details on test material:
- Lot/batch No.: PPA 4357
- Expiration date of the lot/batch: No specific expiry date. At least 10 years or as long as enzyme activity is preserved.
- Stability under test conditions: Solutions in water are stable for at least 24 hours at room temperature or 4 degrees Celsius.
- Storage condition of test material: Refrigerated in the dark.

Test animals

Species:
rat
Strain:
other: CD strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 33-40 days
- Weight at study initiation: Weighed 91 to 123 g seven days after arrival.
- Fasting period before study: None
- Housing: The cages used were Type TRIS made of a stainless steel body measuring 54 x 38 x 20 cm with a stainless steel mesh lid and floor.
- Diet: Ad libitum pelleted rodent diet, RMI(E) SQC.
- Water: Ad libitum. Water, taken from the public supply (Suffolk Water Company, Lowestoft, England}, was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 12 days

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

IN-LIFE DATES: From: 21.07.94 To: 17.10.94

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Remarks:
and purified water for the other control
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test enzyme was supplied in a 24% aqueous solution of monopropylene glycol. It was administered to high dosage animals as received at a volume-dosage of 10 mL/kg bodyweight. For the lower dosages, the test enzyme was diluted with an appropriate amount of purified water (obtained by reverse osmosis) to provide the required dosages at a constant volume-dosage. Vehicle control animals (Group 2) received a 24% aqueous solution of monopropylene glycol in purified water whilst the other control group (Group 1) received purified water only. The volume dosage in each case was 10 mL/kg bodyweight. All formulations were prepared on the day before administration.
VEHICLE
- Concentration in vehicle: 24% aqueous solution of monopropylene glycol.
- 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 . All samples were deep frozen and despatched for analysis. The results of these analyses indicated values approximately 30-65% higher than expected; these were considered to be acceptable as the method for determining the enzyme activity was changed after the initial determination.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Concentration is in mg TOS
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Concentration is in mg TOS
Dose / conc.:
2 600 mg/kg bw/day (nominal)
Remarks:
Concentration is in mg TOS
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. The schedule was: Week 1 daily; Weeks 2-4 twice weekly (middle and end of each week); Weeks 5-13 once each week.

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: Not stated.
- 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
Other examinations:
ORGAN WEIGHTS: Yes.
The weights of adrenals, kidneys, testes and thyroid (with parathyroids) were separately recorded for left and right sides and summed for presentation in the tables of group mean values and appendices of individual values.
Statistics:
The main tests used were analysis of covariance (ANOCOVA), Student's t-test, Fisher's Exact Probability test, Kruskall-Wallace, Mann-Whitney U-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
Description (incidence and severity):
Salivation associated with dosing was observed on a few occasions in isolated animals receiving 2.6 g TOS/kg/day. In view of the very few animals affected this could not, with any confidence, be ascribed to treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female, a control receiving purified water, died during Week 13. There were no significant signs before death. Necropsy or histopathological examination could not establish a possible cause of death in view of extensive cannibalisation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The weight gains of males receiving 0.5 g TOS/kg/day were slightly lower than those of either group of control males but, in the absence of a similar finding among males receiving the highest dosage, this was considered fortuitous.
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):
not specified
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Males receiving 2.6 g TOS/kg/day had slightly low haemoglobin concentrations when compared with both Control groups (p<0.05). Since other erythrocyte characteristics (packed cell volumes, erythrocyte counts and erythrocytic indices) were unaffected and no similar trend was observed in females this could not, with any confidence, be attributed to treatment. Several inter-group differences attained statistical significance when compared with either of the control groups (p<0.05) but these were minor, lacked dosage-relationship or were inconsistent between the sexes and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
After 13 weeks of treatment, liver weights were slightly high, when compared with the control group, in females that received 2.6 g TOS/kg/day. The liver weights of males were clearly unaffected by treatment. In addition, the kidney weights of females that received 2.6 g TOS/kg/day were also slightly high. However, as the female low dose kidney weights were similarly high, this change was considered to have arisen by chance and not as a consequence of treatment.
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:
>= 2 600 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.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It was concluded that the administration of CGT-ase SP 501, batch PPA 4357 at dosages 0.1, 0.5 or 2.6 g TOS/kg/day to CD rats for 13 weeks did not produce any evidence of toxicity.
Executive summary:

Groups of 20 male and 20 female CD rats received CGT-ase SP 501, batch PPA 4357 at 4, 20 or 100% as supplied (equivalent to 0.1, 0.5 or 2.6 g TOS/kg/day) by daily oral gavage administration for 13 weeks. Two similarly constituted control groups received either purified water or a 24% aqueous solution of monopropylene glycol and each served to generate contemporaneous control data.

There were no treatment-related signs. Weight gains, food intakes and food conversion efficiencies were unaffected by treatment. There were no changes in the blood that were related to treatment. Liver weights were slightly high after 13 weeks in females given 2.6 g TOS/kg/day. The incidence of reactive hyperplasia of the mandibular lymph nodes in males was slightly high when compared with the vehicle controls.

It was concluded that the administration of CGT-ase SP 501, batch PPA 4357 at dosages 0.1, 0.5 or 2.6 g TOS/kg/day to CD rats for 13 weeks did not produce any evidence of toxicity.