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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1978
Report date:
1978

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The subacute percutaneous toxicity of Subtilisin was assessed. The systemic and local effects of daily repeated applications of 10 mg/kg/day of the test material Subtilisin, prepared as a 0.5% w/v solution in water and 0.5% w/v in sodium tripolyphosphate buffer, respectively. The application was performed daily for 28 consecutive days without occlusion to the abraded and intact skin of the albino rabbit, an area equal to 10% of the total body surface clipped free of hair. Four female and four male rabbits were used per group, i.e. 32 rabbits in total including two negative control groups.
GLP compliance:
no
Remarks:
The study was performed before GLP was implemented but was performed according to state of the art at that time.
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Subtilisin
EC Number:
232-752-2
EC Name:
Subtilisin
Cas Number:
9014-01-1
Molecular formula:
Not available.
IUPAC Name:
Subtilisin
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.
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:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Substance type: UVCB
- Physical state: Powder
- Lot/batch No.: FPF 312/20
- Expiration date of the lot/batch: No specific expiration 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 Celcius
- Storage condition of test material: 4 degrees of C

Test animals

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Morton Commercial Rabbits, Parsonage Farm, Stanstedt, UK
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 2.2 - 2.7 kg
- Age at study initiation: Young adults, 3 - 3.5 months
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 14-18C
- Humidity : 50% ( range 40-60 %)
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs

Administration / exposure

Type of coverage:
open
Vehicle:
other: water, respectively sodium tripolyphosphate buffer
Details on exposure:
TEST SITE
- Area of exposure: 10% of body surface
- Time intervals for shavings or clipplings: The animals were shaven as needed - no specific interval given in report.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Four hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg
- Concentration (if solution): 0.5% w/v
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0.1 % sodium tripolyphosphate buffer


USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Four hours per day
Frequency of treatment:
Each day for 28 days.
Doses / concentrations
Remarks:
Doses / Concentrations:
10 mg/kg, i.e. 2.0 mL/kg of a 0.5% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal per unit body weight
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on experience from short term dose-range studies, the dose was selected to provide some toxicological effects but still avoiding extreme irritation as an endpoint

Positive control:
No positive control

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Days 1, 2, 3, 7, 14, 21, 28

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No data


OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 4 & 5 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- Parameters checked in table No 6 & 7 were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes ( Appendix 9 in report)
HISTOPATHOLOGY: Yes (Appendix 9 in report)
Other examinations:
Twelve main organs were weighed
Statistics:
Organ weights were evaluated by analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
no effects observed
Description (incidence and severity):
skin reactions were confined to sporadic, minimal grade
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Any effects were confined to slight acanthosis, and a variable, slight infiltration of the underlying dermis by leucocytes
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No clinical effects and no mortality

BODY WEIGHT AND WEIGHT GAIN: Within normal limits during the study.

FOOD CONSUMPTION: Within normal ranges during the study.

HAEMATOLOGY: Within normal ranges, unaffected by treatment.

CLINICAL CHEMISTRY Within normal ranges, unaffected by treatment.

NEUROBEHAVIOUR: Behaviour normal throughout the study

ORGAN WEIGHTS: No differences between groups.

GROSS PATHOLOGY: No treatment related gross lesions present

HISTOPATHOLOGY: NON-NEOPLASTIC There were no treatment related effects other than minor local skin effects. These were slight acanthosis, occasional patchy parakeratosis and variable slight infiltration of underlying dermis by leucocytes.



Effect levels

Key result
Dose descriptor:
conc. level:
Effect level:
> 10 mg/kg bw/day (nominal)
Based on:
dissolved
Remarks:
0.5% w/v solution in water or buffer
Sex:
male/female
Basis for effect level:
other: detailed skin reactions, clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The only effects of 28-day repeated treatment of intact and abraded skin with Subtilisin, buffered and unbuffered, were microscopically detected minor changes at the site of application.
Executive summary:

A percutaneous 28-day repeated application study in rabbits was conducted by Life Science Research (now Huntingdon Life Sciences Ltd.) to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause dermal toxicity. Only one dose was applied daily, 10 mg/kg/day, diluted in either water or buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each group consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed. The study concluded that the test substance, Subtilisin, was without any significant effects and thus may be considered virtually harmless.

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