4-phytase

Endpoint summary

Description of key information

The skin and eye irritation potential of 4 -phytase has been tested and no dermal or eye irritation was observed.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available

Reference 2

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

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

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.

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

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro eye irritation study does not need to be conducted because adequate data from an in vivo eye irritation study are available

Reference 2

Endpoint:

eye 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charles River, D-88353, Germany
- Age at study initiation: not specified
- Weight at study initiation: 3.1 - 5.1 kg
- Housing: Kept individually in PPO- plastic 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: 4 months for first animal and 2 weeks for additional animals

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

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.1 mL of undiluted test item was used (equivalent to 26.51 mg enzyme concentrate dry matter/mL or 15.64 mg active enzyme protein/mL)

Duration of treatment / exposure:

Single exposure

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 females

Details on study design:

REMOVAL OF TEST SUBSTANCE: Test substance was not removed after application to the eye.

SCORING SYSTEM: Draize

TOOL USED TO ASSESS SCORE: At 24 hours after treatment, instillation of Fluorescein followed by flushing with 0.9% NaCl was included. Both eyes were examined again using UV light to detect corneal damage.

Irritation parameter:

cornea opacity score

Basis:

animal: ALL

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal: ALL

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal: ALL

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal: ALL

Time point:

24/48/72 h

Score:

0

Max. score:

3

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No irritation was observed in any of the animals.

Other effects:

No clinical signs were observed in daily observations

Interpretation of results:

GHS criteria not met

Conclusions:

No irritation was observed in any animal tested.

Executive summary:

The objective of this study was to assess the ocular irritation potential of the test substance following OECD Guideline 405.  The initial test, with 0.1 mL of test substance (equivalent to 26.51 mg enzyme concentrate dry matter/mL or 15.64 mg active enzyme protein/mL). applied to the left eye and the right eye as control, was conducted on one rabbit. After 24 hours, fluorescein was instilled and rinsed with 0.9% NaCl. The eye was examined with a UV light to detect corneal damage. The eye was also examined at 48 and 72 hours. Changes in the eye were graded according to a numerical scale 1, 24, 48, and72 hours after treatment. The confirmatory tests were carried out on an additional 2 rabbits and the tests and examinations were performed in a similar manner.

 

No clinical signs were observed in the animals during the daily observations. The mean values for all three rabbits were 0 for cornea opacity, iris lesion, chemosis of conjunctiva, and redness of conjunctiva.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)