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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 1989 - January 1990
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: US FDA
Version / remarks:
1982
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
other: SPF rats of the stock Mol:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: M0llegaard Breeding Center ApS, Ejby, DK-4623 Lille Skensved
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 75-91 g
- Housing: Type III Macrolone cages (42 x 26 x 15 cm) with two in each cage. Males and females were kept separated. Each cage was provided with a wiremesh grid at the bottom.
- Diet: A complete powdered rodent diet "Altromin 1314" was available ad libitum.
- Water: Drinking water acidified with hydrochloric acid to about pH 2.5 was available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 15%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 08.08.1989 to 08.09.1989

Administration / exposure

Route of administration:
oral: feed
Details on route of administration:
The test substance was given incorporated in the diet for at least 28 days, and until autopsy. Control rats received the powdered standard diet onlky.
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:

- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Altromin 1314
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity and stability of the test item was investigated before commencement of dosing. A 5% diet was prepared and six samples taken out and stored at room temperature for 5 days until analysis.

On day 0 six samples were taken from each dietary admixtures to the high and low dose groups for analysis for activity and homogeneity. The six samples were taken from six representatively selected locations in the dietary admixture container. Furthermore one sample from intermediate dose group and control group was taken for analysis of activity.

At the end of week 1 (the morning of day 7), one sample of the three admixtures were taken for analysis of stability. On day 7, 14 and 21, one sample from each of the three admixtures were taken for analysis, and on day 31 another three samples were taken for analysis of stability. After termination of the study, a sample of the test substance was taken for analysis of stability. All samples were stored at 5°C.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: %
Remarks:
% in the diet (w/w)
Dose / conc.:
0.25 other: %
Remarks:
% in the diet (w/w)
Dose / conc.:
1 other: %
Remarks:
% in the diet (w/w)
Dose / conc.:
4 other: %
Remarks:
% in the diet (w/w)
No. of animals per sex per dose:
10
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption was recorded weekly for each cage during the treatment period. The food conversion ratio was calculated group-wise for each sex at weekly intervals.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before commencement of the study and before termination (day 22).
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 23 male rats and day 24 female rats
- Anaesthetic used for blood collection: Yes (carbondioxide)
- Animals fasted: No
- How many animals: all


CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:


URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Possible outliers were identified, too. Thereafter each continuous variable was tested for homogeneity of variance with Bartlett's test. If the variance was homogeneous, possible intergroup differences were assessed with Dunnett's test. If the variance was heterogeneous, each variable was tested for normality by the Shapiro-Wille method. In case of normal distribution, possible intergroup differences were identified with Student's t-test. Otherwise the possible intergroup differences were assessed by Kruskal-Wallis's test. If any significant intergroup differences were detected, the subsequent identification of the groups was carried out with Wilcoxon Rank-Sum test. The food conversion ratio data were analyzed with Least-Square's Means Test. The statistical analyses were made with SAS-Procedures.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Piloerection was observed in one animal after blood sampling on day 23.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weight of males in group 3 on day 14 was statistically significantly lower than the mean values of the control group. Apart from this change, the mean body weight of the low and intermediate dose groups (group 2 and 3) were comparable throughout the acclimatization and the dosing period to those of the control group. The mean body weight in the dosing period of the high dose group (group 4) was slightly, but not statistically significantly, increased for both sexes, compared to that of the control group.
Body weight gain: The body weight gain of both sexes of group 4 was slightly higher than that of the control group during the dosing period. The body weight gain of groups 2 and 3 was comparable to that of the control group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Throughout the dosing period, the food consumption of each of the low and intermediate dose groups (group 2 and 3) were comparable to those of the control group. The food consumption in the high dose group (group 4) was throughout the dosing period somewhat higher for both sexes compared to the controls, but only statistically significantly higher for females in weeks 2 and 3.
Food efficiency:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hemoglobin (Hb) was statistically significantly decreased in females of group 4 (p < 0.05). All individual data in this group and the control group are within the range of historical values with the exception of the following: Two values in the control group were high and out of range of the historical values and one value in group 4 (females) was low and also out of range. For this reason, the low value of hemoglobin in females of group 4 is considered to be without biological significance. The number of white blood cells (WBC) was statistically significantly higher in females of group 2 (p <0.05). Five values in this group were high and out of range of historical values. The level of the control values were also high, but within the range of historical value. Since no dose dependency is present, the high number of white blood cells in females of group 2 is considered to be incidental.
n males of groups 2 and 3, the number of monocytes (MONO) was statistically significantly decreased compared to the value of the control group (p < 0.05). This deviation is considered to be of no importance.
Apart from these deviations in the hematological values, all other mean values of the treated groups were comparable to the control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In male rats, serum carbamide (UREA) was statistically significantly lower in groups 3 and 4, and total protein (PROTEIN) was statistically significantly lowt;r in groups 2 and 4 (p < 0.05). In female rats of group 3 and 4 serum carbamide had also decreased (p < 0.05), but these values were not statistically significantly lower compared to the controls. All individual serum carbamide data in groups 3 and 4 (males) were within the range of historical values. Since an adverse effect of a treatment in general is followed by an increase in serum carbamide values, the relatively low serum carbamide values are considered of minor importance. The level of total protein values in male rats of all groups were low compared to the historical values. Two values in the control group were out of range of the historical values, whereas 7, 4 and 10 values were out of range in groups 2, 3 and 4, respectively. There were 10 males per group. Since a clear dose-response effect is not present, it is questionable, whether the lowering in total protein represents an effect of the treatment. ASAT had decreased in males of group 4 and was statistically significantly lower in females of group 3 and 4 (p < 0.05). All individual data in females of groups 3 and 4 are within the range of historical values. For this reason and since an adverse effect of a treatment in general is followed by an increase in ASAT, the low values in those groups are considered to be without toxicological significance.
Gamma globulin had increased in female rats of group 4 (p < 0.05). This finding is considered to be an unspecific immunological reaction. Apart from the above mentioned, all mean values of the treated groups were comparable to the control group, with the exception of a decrease of glucose and creatinine in females of group 3 (p < 0.05). These findings are most likely incidental and considered of no importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were very few gross pathological findings. They are all well known incidental findings in this rat stock.
Petechial hemorrhages in thymus were observed in treatment but not control groups and are probably agonal hemorrhages caused by asphyxia at sacrifice. It is a coincidence that no such changes were found in rats in the control group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
All histopathological findings are incidental findings most of which are well known in the present rat stock.

Those not well known are hepatic cirrhotic focus, cortical tubules in renal marrow (probably malformation), and cerebral cortical cyst (probably malformation). They appeared with an incidence of only one.

Renal focal interstitial mononuclear cell accumulation, accumulation of plasma cells in mesenteric lymph node, and focal thyroiditis appeared in group 4, but none of those changes can be realted to treatment.
Histopathological findings: neoplastic:
not specified
Other effects:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 4 other: %
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
food efficiency
ophthalmological examination
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
It is concluded that the no effect level of beta-glucosidase batch DCN 0005 to rats dosed for 4 weeks up to a level of 0.25% in the diet was without any effect. At a dosage of 1 and 4% in the diet only minor lowerings in serum carbamide and ASAT were registered, and therefore the high dosage (4%) can be considered as the no ill effect level.

The substance is not classified with regards to systemic toxicity according to the CLP Regulation.
Executive summary:

The present study was undertaken in order to evaluate the toxicity of beta-glucosidase batch DCN 0005 in the rat, while incorporated in the diet for four weeks. Three groups each of twenty rats (10 males and 10 females) received beta-glucosidase batch DCN 0005. The dose levels were 0.25, 1 and 4 % (w/w) in the diet. A control group similarly constituted was given the standard diet only. Throughout the study clinical signs of reaction to the treatment were recorded. Body weights and food consumption were measured weekly. The rats were subjected to ophthalmoscopic examination. Before termination, blood samples were taken from all animals for hematology and blood chemistry. At the end of the treatment period all rats were killed and subject to detailed pathology. A number of organs were weighed. Specimens of a list of organs and tissues were fixed. Specified organs and tissues were examined microscopically.

Throughout the dosing period there were no clinical symptoms of adverse reaction to the treatment. Throughout the dosing period, the food consumption in the high dose group (4% in the diet) was higher for both sexes compared to the controls, but only statistically significantly higher for females in weeks 2 and 3. Throughout the dosing period, the food conversion ratio of the treated groups was comparable to that of the control group. The mean body weight and body weight gain in the dosing period of the high dose group was slightly, but not statistically significantly increased for both sexes. The eyes of the rats were not affected by the treatment. Hematological examination showed a decrease of hemoglobin in female rats in the high dose group. Total protein was lower in male rats of the low and high dose groups, and serum carbamide had decreased in male rats of the intermediate and high dose groups. ASAT was lower in female rats of the intermediate and high dose groups. Gamma globulin had increased in female rats of the high dose group. As all ASAT and serum carbamide values are within range of historical data and as an adverse effect is expected to be followed by an increase, the decreased ASAT and serum carbamide values are considered to be of no toxicological importance. The deviations in total protein and gammaglobulin values are not considered to be toxic effects of the treatment. The absolute and relative organ weights of the treated groups were comparable to those of the control group. At the gross pathological and histopathological examination no treatment-related findings were found.

It is concluded that no effect level of beta-glucosidase batch DCN 0005 dosed to rats for 4 weeks up to a level of 0.25% in the diet was without any effect. At a dosage of 1 and 4% in the diet only minor lowerings in serum carbamide and ASAT were registered, and therefore the high dosage (4%) can be considered as the no ill effect level.