4-phytase

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme of 4-phytase (CAS 9001-89-2, IUBMB 3.1.3.26)

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

Active enzyme of 4-phytase (CAS 9001-89-2, IUBMB 3.1.3.26)

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:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

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 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:

liquid

Details on test material:

% Total Organic Solids: 25.27%

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, D-88353 Kisslegg, Germany
- Age at study initiation: not specified
- Weight at study initiation: 3.5-3.7 kg
- Housing: Individually kept in PPO-plast cages (floor area: 4200 cm2) with perforated floor. The tray under the floor was cleaned twice per week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approximately 2 months

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21°C ± 3°C
- Humidity (%): 55% ±°15%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark

Test system

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

other: The fourth quadrant of the preliminary test was used as a negative control

Amount / concentration applied:

0.5 mL (which is equivalent to 132.51 mg enzyme concentrate dry matter/mL or 78.19 mg active enzyme protein/mL)

Duration of treatment / exposure:

Preliminary Test: 3 minutes, 1 hour, 4 hours
Confirmatory Test: 4 hours

Observation period:

72 hours; one animal was further observed on Day 8

Number of animals:

3

Details on study design:

TEST SITE
- Area of exposure: 2.5 x 2.5 cm
- Type of wrap if used: surgical gauze patch to cover the test area. Each gauze patch was fixed to the site with Scanpor tape. Nobafix gauze was loosely wound the trunk to secure the dressing.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): lukewarm water and mild soap
- Time after start of exposure: Initial Test: 3 minutes

OBSERVATION TIME POINTS: Immediately after patch removal, 1 hour, 24, 48, and 72 hours

SCORING SYSTEM: See another information on materials and methods for scoring scale.
- Method of calculation: The animals were graded on a numerical scale for the 4 hour exposure time based on 3 readings (24, 48, and 72 hours) for both erythema and edema. The scores were then divided by 3 to obtain to obtain Individual Mean Scores for each rabbit. The mean score erythema and edema for the three rabbits was calculated from the Individual Mean Scores.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

erythema score

Basis:

other: mean score of all animals

Time point:

24/48/72 h

Score:

0.8

Max. score:

4

Reversibility:

fully reversible within: 8 days for initial animal and 48 hours for other 2 animals

Results 2

Irritation parameter:

edema score

Basis:

other: mean score of all animals

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

Initial Test
No clinical signs were observed in the animal during daily observations. No oedema was observed at the test or control sites at any of the examinations at termination of exposure, and neither at the examinations at 1, 24, 48, or 72 hours after exposure termination. Very slight erythema was observed at the 4-hour exposure test site at 1, 24, 48, and 72 hours after exposure. No reactions were observed 8 days after treatment. For the 4- hour test site, the Individual Mean Score calculated using only the scores from the 24, 48, and 72 hour observations points were 1.0 and 0.0 for erythema and oedema, respectively.

Confirmatory Test
No clinical signs were observed in the animals during daily observations. No oedema was observed at the test or control sites at any of the examinations at termination of exposure, and neither at the examinations at 1, 24, 48, or 72 hours after exposure termination. Very slight erythema was observed in both animals. One animal had very slight erythema at the readings 24 and 48 hours after exposure and the other had very slight erythema 1, 24, and 48 hours after exposure. The Individual Mean Scores for erythema and oedema for both of the remaining animals were 0.7 and 0.0, respectively.

The Individual Mean Scores were combined to give an overall Mean Score for the test item, which were 0.8 and 0.0 for erythema and oedema, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The overall test substance Mean Scores (all three animals) was 0.8 and 0.0 for erythema and oedema, respectively.

Executive summary:

The objective of the study was to evaluate local irritant effects of the test substance to rabbit skin following a single topical exposure. The study was conducted according to OECD Guideline 404 and evaluated by the Commission Directive 2001/59/EC of 6 August 2001 adapting to technical for the 28th time Council Directive 67/548/ECC.

 

The initial test was carried out on one rabbit with the back divided into 4 quadrants. The sites were used to examine the test substance at 3 minutes, 1 hour, and 4-hour exposure times and the last quadrant was a negative control. The animal was observed and scored at 1, 24, 48, and 72 hours after exposure. Very slight erythema (barely perceptible) was observed at the 4-hour test site at the readings 1 hour, 24, 48, and 72 hours after termination of exposure. No oedema was observed.

 

A confirmatory test was carried out concurrently with two additional rabbits with only a 4-hour exposure site (0.5 mL per test site which is equivalent to 132.51 mg enzyme concentrate dry matter/mL or 78.19 mg active enzyme protein/mL) and a negative control. The animals were observed and scored 1, 24, 48, and 72 hours. Very slight erythema (barely perceptible) was observed in one animal at 24 and 48 hours after termination of exposure and at 1, 24, and 48 hours in the second animal. No oedema was observed. No clinical signs were observed in any of the animals during the daily observations.

 

The overall test substance Mean Scores (all three animals) was 0.8 and 0.0 for erythema and oedema, respectively.