Subtilisin

Repeated dose toxicity: dermal

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

Test material information

Constituent 1

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

Details on test material:

- 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.

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 - 2.8 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 clippings: 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
8.8 mg enzyme concentrate dry matter/kg

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: all animals (32)
- Parameters checked: hemoglobin concentration (Hb), Erythrocyte count (RBC), Leucocyte count, total (WBC), neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and packed cell volume (PCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: All animals (32)
- Parameters checked: Urea concentration, glucose concentration, total protein concentration, electrophoretic protein fractions, alkaline phosphatase activity (SAP), glutamate-pyruvate transaminase activity (SGPT), glutamate-oxalacetate transaminase activity (SGOT)

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. The observed responses were considered to be related to the daily mechanical handling and material application.

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

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:

> 1.4 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.