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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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

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 Alpha-Amylase was assessed. The systemic and local effects of daily repeated applications of 6 KNU (Alpha-Amylase enzyme activity units)/kg/day of the test material, prepared as a 0.62% w/v solution in water and 0.62% 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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Termamyl® (SP 95), batch ATE 020
- Substance type: UVCB
- Physical state: Brown powder
- Lot/batch No.: ATE 020
- 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 and environmental conditions:
- Source: Outbred New Zealand White rabbits, breeder not listed in report
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 1.97 - 2.84 kg
- Age at study initiation: Young adults, 2.5 - 3 months
- Diet (e.g. ad libitum): Standard diet (Diet RAF from Labsure Animal Foods, Poole, Dorset) ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 13-17
- 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): 2 mL/kg/day, corresponding to an amylase activity of 6 KNU/kg/day
- Concentration (if solution): 0.62% 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): 0.3 % sodium tripolyphosphate buffer


USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application to prevent ingestion of any test material.
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
Dose / conc.:
12.4 other: mg/kg bw
Remarks:
Doses / Concentrations:
12.4 mg/kg, i.e. 2.0 mL/kg of a 0.62% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on experience, the dose was selected to avoid 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: Daily, just before dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment period

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined at weekly intervals throughout the treatment period and mean daily diet consumption calculated as g food/kg body weight/day.

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
- The following parameters were examined: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- The following parameters were examined: urea, glucose, albumin, total protein, electrophoretic protein fractions, alkaline phosphatase, glutamate-pyruvate transaminase activity, glutamate-oxalacetate transaminase activity

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
HISTOPATHOLOGY: Yes (all animals - 20 organs/tissues examined in all groups)
Other examinations:
Eleven main organs were weighed
Statistics:
Organ weights were evaluated by analysis of variance.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Two animals died during the study from unrelated causes as determined at necrosis
Dermal irritation:
no effects observed
Description (incidence and severity):
Skin reactions were confined to erythematous responses at the site of application, however no oedema.
Mortality:
no mortality observed
Description (incidence):
Two animals died during the study from unrelated causes as determined at necrosis
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):
no effects observed
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 mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No clinical effects. Two animals died during the study from unrelated causes as determined at necrosis.

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 effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.



Effect levels

Key result
Dose descriptor:
conc. level:
Effect level:
> 12.4 mg/kg bw/day (nominal)
Based on:
dissolved
Remarks:
0.62% 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;

Applicant's summary and conclusion

Conclusions:
The only effects of 28-day repeated treatment of intact and abraded skin with alpha-amylase, buffered and unbuffered, were minor effects locally at the site of application without any initiation of any detectable systemic response. The local skin effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin. The local irritation effects could easily be caused by the content of smaller amounts of protease enzyme activity in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such side activities.
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, alpha-amylase (batch ATE 020), to cause dermal toxicity. Only one dose was applied daily, 12.4 mg/kg/day, diluted in water respectively buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each of the four groups applied consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles were the same and state of the art was followed. The study concluded that the test substance, alpha-amylase, was with only minor effects locally at the site of application without initiation of any systemic effects. The local effects seen at the site of application could easily be caused by the content of smaller amounts of protease enzyme activity in the test material, which was in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such protease side activities.