Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February - 19 May, 1981
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1981
Report Date:
1981

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1981
Deviations:
no
Principles of method if other than guideline:
Guideline OECD 408 was adopted for the first time in 1981, at the time where the present study was performed. The principles of the new guideline was however followed. The current version of OECD 408, revised in 1998, places additional emphasis on neurotoxic, immunological and reproductive organ effects as well as the need for more careful clinical observations of the animals compared to the 1981 version. These changes are not considered to be important for toxicological evaluation of enzymes produced by non-toxigenic, non-pathogenic strains for which the main safety issue of concern is respiratory allergy and in some cases primary irritation. This is supported by numerous 13-week oral toxicity studies performed on enzymes according to OECD 408 (1998), in which no effects on the additional parameters have been reported.
Therefore the present study is considered suitable for the evaluation of sub chronic toxicity of subtilisin.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Substance type: UVCB
- Physical state: powder
- Lot/batch No.: PPA 1180
- Expiration date of the lot/batch: 1991 (default expiration time is 10 years, but the batch has not expired, as long as the enzyme activity is preserved)
- Stability under test conditions: Solutions in water are stable for at least 24 hours at room temperature or 4 degrees Celcius
- Storage condition of test material: 4 degrees of C

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Weight at study initiation: 60-80 g
- Fasting period before study: None
- Housing: 3 animals per cage, separate sex
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 50% ( range 26-80 %)
- Air changes (per hr): 14
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1981-02-16 To: 1981-05-19

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Distilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 16, 40 and 100 mg/mL, corresponding to 14, 36 and 89 mg TOS/mL
- Amount of vehicle (if gavage): constant volume 10 mL/kg b.w.
- Purity: distilled water
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 16, 40 and 100 mg/mL, corresponding to 0, 14, 36 and 89 mg TOS/mL
Basis:
nominal in water
No. of animals per sex per dose:
15
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on experience from dose-range studies, the top dose was selected to provide some toxicological effects but still avoiding death as an endpoint


Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early morning and late afternoon each day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and further more detailed examination of each animal once a week

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

WATER CONSUMPTION : Yes but only by visual assessment of water intake

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 6 and week 13
- Anaesthetic used for blood collection: Yes (ether anaesthesia)
- Animals fasted: No
- How many animals: 10 males and 10 females from negative control groups and high dose groups
- Parameters checked: Red blood cell count (RBC), White blood cell count (WBC), Packed cell volume (PCV), Haemoglobin conc (Hb), Differential leucocyte count, Platelet count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 6 and week 13
- Animals fasted: No
- How many animals: 10 males and 10 females from negative control groups and high dose groups
- Parameters checked: Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, lactate dehydrogenase, blood urea nitrogen, Glucose, Sodium (Na), Potassium (K), Protein (total), Chloride (Cl), Cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 6 and week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Deprivation of feed and water during the 4-hour sampling period
- Parameters checked: Glucose, volume, protein, ketones, urobilinogen, colour, appearance, pH, specific gravity, microscopic examination of the spun deposit.

NEUROBEHAVIOURAL EXAMINATION: No data
The animals were observed daily in accordance with state of the art in 1981

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes.
The majority of the 16 premature decedents were found dead with red staining around nose and mouth. Most decedents showed red patches and oedema of the lungs. Three animals had haemorrhagic tracts or holes in the left lung lobe on post mortem examinination, consistent with lung damage as a result of gavage dosing. Both control and treated rats showed hyperplasia of the submandibular lymph nodes. This could be due to a previous sialodacryoadenitis virus infection. None of the lesions were attributed to toxic effects of the test material.
HISTOPATHOLOGY: Yes. Mild and focal alveolitis was identified in both control and treated animals - the cause was not identified. One animal showed a focal chronic alveolitis. Foreign material was identified in the alveolar air spaces of this animal, suggesting that the cause was inhalation or misdosing of foreign test material.
Other examinations:
FAECAL ANALYSIS: Yes
- Time schedule for collection of faeces: Week 6 and week 13
- Metabolism cages used for collection: Yes
- Animals fasted: Deprivation of feed and water during the 4-hour sampling period
- Parameters presented in appendix 12, 13 and 14 were examined.

Weight of individual organs: yes
- Time schedule for collection of organs: At necropsy
- Results presented in table 12 and 13 .
Statistics:
Organ weight and body weight data were statically analysed by computerized analysis of variance incorporating t-test. The level of probability chosen as significant was p<0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
There were 16 premature deaths. Within males: 8 animals in the top dose group, 1 in mid dose group. Within female: Two animals in the top and mid groups, and 3 animals in the low dose group.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were 16 premature deaths. Within males: 8 animals in the top dose group, 1 in mid dose group. Within female: Two animals in the top and mid groups, and 3 animals in the low dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights were significantly reduced at the highest dose level in males.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
There was a slight dose-related reduction in food consumption in males. Food consumption in females was lower than controls but without dose-relation.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no haematological changes considered to be related to treatment.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Urea nitrogen (BUN) and alanine aminotransferase (ALT) were significantly reduced at the highest dose level in males.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no abnormalities considered to be related to treatment.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The changes that were identified were not considered to of toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No lesions were seen at finalization of the study which could be attributed to toxic effects of the dosed test material. Many of the 16 premature deaths showed findings in the lungs from damage due to gavage dosing or unintended dosing of test material in
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
Details on results:
CLINICAL SIGNS AND MORTALITY: The major clinical observation in this study was increased difficulty of dosing animals receiving 1000 mg/kg bw/day ~ 891 mg TOS/kg bw/day. This difficulty, resulting from struggling during dosing, caused many of the 16 premature deaths due to aspiration. When using oral gavage dosing, the dose volume of 10 ml/kg body weight can easily lead to unintended exposure by inhalation because test substance is deposited in the pharyngeal cavity after reflux from the stomach. This is a likely event in struggling animals, but is as such an artifact caused by the way of dosing the animals.

BODY WEIGHT AND WEIGHT GAIN: : Body weights were significantly reduced at the highest dose level in males.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY: There were no haematological changes considered to be related to treatment.

CLINICAL CHEMISTRY: Clinical Urea nitrogen (BUN) and alanine aminotransferase (ALT) were significantly reduced at the highest dose level in males.
The reduced clinical chemical parameters BUN and ALT could be indicators of lever damage but due to lack of histopathological changes in the lever of these animals, this change is considered of no toxicological significance.


URINALYSIS

NEUROBEHAVIOUR

ORGAN WEIGHTS: The top dose males showed reduced absolute liver and spleen weights with no concurrent change in relative weights, unaltered absolute but increased relative brain weight, increased absolute and relative adrenal weights - could be the result of the difficulty in dosing (see reference 1 and 2 below). The males receiving 400 and 1000 mg/kg bw/day also showed increased relative lung weights, but this could be due to reduction in body weight.
References:
1. Scharer, K. (1977). The effect of the chronic underfeeding on organ weights of rats. Toxicology, 7: 45-46.
2. Oishi, S., Oishi H. and Hiraga, K. (1979). The effect of food restriction for 4 weeks on common toxicity parameters in male rats. Toxicol. Appl. Pharmacol., 47: 15-22.
The absolute and relative thyroid weights were found to be significantly increased in all treated males, but this finding is not judged to be of great significance for the following reasons:
1) The thyroid gland is small in rats with a color similar to the surrounding tissue and very difficult to dissect precisely for weighing. This is also indicated by the coefficient of variability (CV = 100 x SD/mean weight) which can be calculated from the thyroid weight data in table 12. The CV is above 20% in all groups and in the female control group, it is even 55%, showing a very high variability in these data.
2) The histopathology did not reveal any changes.
3) Historical data showed that the seen increase in weight was possibly due to unusual low thyroid weight data in the control animals for both males and females.
4) The present study was finalized Sept. 1981, and reports covering 13 week oral toxicity studies in rats from the same CRO in the following year, 1982 and onwards, does no longer include the thyroid weight as a parameter. This decision of excluding the organ weight of the thyroids is most likely taken because these data do not provide any added value. Further, the OECD test guideline 408, 'Subchronic Oral Toxicity - Rodent: 90-Day Study', which was adopted 12 May 1981, has supported this decision as weighing of the thyroid gland was not required in this guideline.


GROSS PATHOLOGY: The effects in the lungs (irregular brown areas) are considered artifacts originating from dosing by gavage especially in case of the higher dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC : The effects in the lungs (brown pigments) are considered originating from dosing by gavage especially in case of the higher dose level.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
> 360 - < 891 mg/kg bw/day (nominal)
Based on:
other: TOS
Sex:
male/female
Basis for effect level:
other: body weight: Slight reduction; food consumption: Slight reduction

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

For details, please see the attached report (scanned pdf file as it is written before electronic files were used).

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) in rats is 400 mg of the subtilisin batch/kg bw/day equivalent to 360 mg Total Organic Solids (TOS)/kg bw/day.
Executive summary:

Guideline OECD 408 was adopted for the first time in 1981, at the time where the present study was performed. The principles of this OECD guideline were, however, followed and the study performed in compliance with GLP. The current version of OECD 408, revised in 1998, places additional emphasis on neurotoxic, immunological and reproductive organ effects as well as the need for more careful clinical observations of the animals compared to the 1981 version. These changes are not considered to be important for toxicological evaluation of enzymes produced by non-toxigenic, non-pathogenic strains for which the main safety issue of concern is respiratory allergy and in some cases primary irritation. This is supported by several 13-week oral toxicity studies performed on enzymes according to OECD 408 (1998), in which no effects on the additional parameters have been reported. The present study is therefore considered suitable for the evaluation of sub chronic toxicity of subtilisin.

 

In conclusion, oral administration (by gavage) of subtilisin to rats at the highest dose level, 1000 mg subtilisin batch/kg bw/day, equivalent to 809 mg Total Organic Solids (TOS)/kg bw/dayfor thirteen weeks, resulted in significantly reduced body weights in males and remarkable struggling against the gavage. Apart from these effects, dosing did not produce any toxicologically significant changes. Consequently, the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the intermediate dose level administered, equivalent to 400 mg of the subtilisin batch/kg bw/day or 360 mg Total Organic Solids (TOS)/kg bw/day.