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Administrative data

Description of key information

The skin and eye irritation potential of xylanase 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

Referenceopen allclose all

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
Endpoint:
skin irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 9, 2013 to March 25, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Version / remarks:
2002
Deviations:
yes
Remarks:
The protocol requires that a single patch be applied to one animal and dose site be scored immediately after the 4-hour patch removal. Due to an oversight evaluation was also completed for 2nd and 3rd animal dosed. This has no impact on the study outcome.
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Received from Robinson Services, Inc. Mocksville or Clemmons, NC on January 4 and 16, 2013.
- Age at study initiation: Young adult
- Housing: The animals were singly housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g. toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet (e.g. ad libitum): Harlan Teklad Global High Fiber Rabbit Diet® #2031. A designated amount of the diet was available to each rabbit (approximately 150 grams/day).
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum by an automatic water dispensing system.
- Acclimation period: 6 or 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-20ºC
- Humidity: 14-37%
- Photoperiod: 12-hour light/dark cycle

Type of coverage:
semiocclusive
Preparation of test site:
clipped
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5mL of the test substance
Duration of treatment / exposure:
4 hours
Observation period:
Individual dose sites were scored according to the Draize scoring system immediately following patch removal, all animals were scored immediately after patch removal (small deviation, see below study design) and at approximately 30-60 minutes, 24, 48, and 72 hours after patch removal.
Number of animals:
3
Details on study design:
-Preparation and Selection of Animals:
Approximately 24 hours prior to application, the animals were prepared by clipping the dorsal area and the trunk. On the day of dosing, but prior to application, the animals were examined for health and the skin checked for any abnormalities. Three healthy naive animals (not previously tested) without pre-existing skin irritation were selected for test. Initially, only one rabbit was placed on test. Since no dermal irritation was observed at the dose site on this animal, the test was completed with two additional rabbits.

-Application of Test Substance:
0.5mL of the test substance was applied to one 6-cm2 intact dose site on each animal and covered with a 1-inch x 1-inch, 4-ply gauze pad. The pad and entire trunk of each animal were then wrapped with semi-occlusive 3-inch Micropore tape to avoid dislocation of the pad. Elizabethan collars were placed on each rabbit and they were returned to their designated cages. No other substances were tested on these rabbits. After 4 hours of exposure to the test substance, the pads and collars were removed and the test sites were gently cleansed of any residual test substance.

-Evaluation of Test Sites/Classification Scheme:
Individual dose sites were scored according to the Draize scoring system immediately following patch removal, all animals were scored immediately after patch removal and at approximately 30-60 minutes, 24, 48, and 72 hours after patch removal. The protocol requires that a single patch be applied to one animal and the dose site be scored immediately after the 4-hour patch removal. Due to a scientist oversight the evaluation was also completed for the second and third animal dosed. This deviation had no impact on the outcome of the study.
Irritation parameter:
erythema score
Basis:
animal: 1, 2, 3
Time point:
other: Mean of 24-, 48-, and 72-hour dermal responses
Score:
0
Max. score:
0
Irritation parameter:
edema score
Basis:
animal: 1, 2, 3
Time point:
other: Mean of 24-, 48-, and 72-hour dermal responses
Score:
0
Max. score:
0
Irritant / corrosive response data:
There was no dermal irritation observed at any treated dose site during the study.
Other effects:
All animals appeared active and healthy and gained body weight during the study. There were no clinical signs observed.

 TABLE 1: INDIVIDUAL BODY WEIGHTS

Animal No.

Sex

Initial

Terminal

3501

F

2071

2161

3502

F

2068

2220

3503

F

2376

2448

 

TABLE 2: INDIVIDUAL SKIN IRRITATION SCORES (ERYTHEMA/EDEMA)

Animal No.

Sex

Time After Patch Removal

Immediately after patch removal

30-60 mins

24 hrs

48 hrs

72 hrs

3501

F

0/0

0/0

0/0

0/0

0/0

3502

F

0/0

0/0

0/0

0/0

0/0

3503

F

0/0

0/0

0/0

0/0

0/0

TABLE 3: SUMMARY OF MEAN SCORES FOR DERMAL RESPONSES

RABBIT NUMBER

ERYTHEMA

EDEMA

3501

0.0

0.0

3502

0.0

0.0

3503

0.0

0.0

Calculated from the 24-, 48-, and 72-hour dermal responses (EEC/GHS)

Interpretation of results:
GHS criteria not met
Conclusions:
Xylanase should not be classified as a skin-irritant.
Executive summary:

A primary skin irritation test was conducted with rabbits to determine the local skin irritant effect of the test substance. This study was conducted according to the method recommended in the OECD Guideline No. 404, April 2002.

Approximately 24 hours prior to application, all animals were prepared by clipping the dorsal area and the trunk. 0.5mL was applied to one 6 cm2 intact dose site and covered with 1 inch x 1 inch, 4-ply gauze pad. The pad and entire trunk of each animal were then wrapped with semi-occlusive tape. Collars were placed on each rabbit. After 4 hours of exposure, the pads and collars were removed and the test sites were cleansed of any residual test substance. The grade of skin reaction was scored according to the Draize’s scoring system immediately following patch removal and at 30-60 minutes, 24, 48 and 72 hours after patch removal. This study was conducted in a stepwise manner. Initially, 0.5mL of the test substance was applied to the skin of one rabbit for 4 hours. Since no dermal irritation was noted in this rabbit, a confirmatory test was completed with two additional healthy rabbits. No dermal irritation was observed at any dose site during the study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

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
Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 9, 2013 to March 25, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other:
Remarks:
The study was performed early in 2013 in compliance with GLP standards and according to OECD Guidelines No. 405 (2002). An updated OECD 405 was available late in 2012. However, this study was part of a whole package of studies for xylanase (90 days oral, Ames test, Chromosomal aberration, Eye & Skin Irritation) initiated and respective protocols approved prior to that date. Additionally, the two versions of OECD 405 differ only in the use of anaesthesia and are similar in the use of sequential approach (one animal at the time). The eye irritation classification of xylanase as determined using OECD 405 (2002) is non-irritant, which will not change using OECD 405 (2012).
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Received from Robinson Services, Inc. Mocksville or Clemmons, NC on January 4, 2013.
- Age at study initiation: Young adult
- Housing: The animals were singly housed in suspended stainless steel caging with mesh floors which conform to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Enrichment (e.g. toy) was placed in each cage. Litter paper was placed beneath the cage and was changed at least three times per week.
- Diet (e.g. ad libitum): Harlan Teklad Global High Fiber Rabbit Diet® #2031. A designated amount of the diet was available to each rabbit (approximately 150 grams/day).
- Water (e.g. ad libitum): Filtered tap water was supplied ad libitum by an automatic water dispensing system.
- Acclimation period: 17 or 24 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-21°C
- Humidity: 15-47%
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
0.1mL of the test substance
Duration of treatment / exposure:
The upper and lower lids were then gently held together for about one second before releasing
Observation period (in vivo):
Ocular irritation was evaluated using a high-intensity white light at 1, 24, 48, and 72 hours post-instillation
Number of animals or in vitro replicates:
3
Details on study design:
-Preparation and Selection of Animals:
Prior to test initiation, both eyes of a group of animals were examined using a white light source and a fluorescein dye procedure. One drop of 2% ophthalmic fluorescein sodium was instilled into both eyes of each rabbit. The eyes were rinsed with physiological saline (0.9% NaCl) after instillation of the fluorescein and then evaluated for corneal damage using an ultraviolet light source. Prior to test substance instillation, the eyes were re-examined and scored for abnormalities according to the "Scale for Scoring Ocular Lesions". Three healthy naive animals (not previously tested) without pre-existing ocular irritation were selected for test. Initially, only one rabbit was placed on test. Since there was no irritation observed in this animal, the test was completed on two additional rabbits to confirm the results.

-Instillation:
Prior to instillation, two to three drops of ocular anesthetic (Tetracaine Hydrochloride Ophthalmic Solution USP, 0.5%) were placed into both the treated and control eye of each animal. 0.1mL of the test substance was then instilled into the conjunctival sac of the right eye of each rabbit by pulling the lower lid away from the eyeball. The upper and lower lids were then gently held together for about one second before releasing to minimize loss of the test substance. The other eye of each rabbit remained untreated with the test substance and served as a control. The rabbits were then returned to their designated cage.All animals were administered 0.33 mL subcutaneous injection of a systemic analgesic (Buprenorphine Hydrochloride i.e.: Buprenex®) at least 30 minutes prior to test substance instillation and at the 24 hour scoring interval to prevent potential discomfort.

-Ocular Scoring:
Ocular irritation was evaluated using a high-intensity white light in accordance with Draize et al. at 1, 24, 48, and 72 hours post-instillation. The fluorescein dye evaluation procedure was used in the treated eye at 24 hours to verify the absence of corneal damage. Individual scores were recorded for each animal. In addition to observations of the cornea, iris and conjunctivae, any other observed lesions were noted.
Irritation parameter:
cornea opacity score
Basis:
animal: 1, 2, 3
Time point:
other: mean of 24-, 48-, and 72-hour scores
Score:
0
Max. score:
0
Irritation parameter:
iris score
Basis:
animal: 1, 2, 3
Time point:
other: mean of 24-, 48-, and 72-hour scores
Score:
0
Max. score:
0
Irritation parameter:
conjunctivae score
Basis:
animal: 1, 2, 3
Time point:
other: mean of 24-, 48-, and 72-hour scores
Score:
0
Max. score:
0
Irritation parameter:
chemosis score
Basis:
animal: 1, 2, 3
Time point:
other: mean of 24-, 48-, and 72-hour scores
Score:
0
Max. score:
0
Irritant / corrosive response data:
No ocular irritation was observed in any treated eye during the study.
Other effects:
All animals appeared active and healthy and gained body weight during the study. There were no clinical signs observed.

TABLE 1: INDIVIDUAL BODY WEIGHTS

Animal No.

Sex

Body Weight (g)

 

 

Initial

Terminal

3401

F

2383

2450

3402

F

2480

2524

3403

F

2233

2364

TABLE 2: INDIVIDUAL SCORES FOR OCULAR IRRITATION

Rabbit No.: 3401 (Female) Rabbit No.: 3402 (Female) Rabbit No.: 3403 (Female)
Hours Hours Hours
1 24 48 72 1 24 48 72 1 24 48 72
 I. Cornea                                
 A. Opacity   0 0 0 0 0 0 0 0 0 0 0 0
 B. Area   4 4 4 4 4 4 4 4 4 4 4 4
 II. Iris                                
 A. Values   0 0 0 0 0 0 0 0 0 0 0 0
 III. Conjunctivae                                
 A. Redness   0 0 0 0 0 0 0 0 0 0 0 0
 B. Chemosis   0 0 0 0 0 0 0 0 0 0 0 0
 C. Discharge   0 0 0 0 0 0 0 0 0 0 0 0

TABLE 3: MEAN SCORES FOR INDIVIDUAL RABBITS

Number

Corneal

Opacity

Iritisa

Conjunctival

Rednessa

Conjunctival

Chemosis

3401

0.0

0.0

0.0

0.0

3402

0.0

0.0

0.0

0.0

3403

0.0

0.0

0.0

0.0

Calculated from the 24-, 48-, and 72-hour scores (EEC/GHS).

Interpretation of results:
GHS criteria not met
Conclusions:
Xylanase should not be classified as an eye-irritant.
Executive summary:

The objective of this study was to assess the ocular irritation potential of the test substance. This study was conducted according to the method recommended in the OECD Guideline No. 405, 24 April 2002.

The study was conducted in a stepwise fashion. Initially, 0.1mL of the test substance was instilled into the conjunctival sac of the right eye of one rabbit. The left eye remained untreated and served as control. The grade of ocular reaction was recorded at 1, 24, 48 and 72 hours post instillation and evaluated by the method of Draize et al. At the 24-hour reading, fluorescein was instilled and then rinsed with 0.9% NaCl. The eye was then examined with an UV-light to detect corneal damage. Since there was no significant ocular irritation noted in this rabbit, the test was repeated on two additional rabbits by the same procedure. No ocular irritation was noted in all three rabbits.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The skin and eye irritation potential of xylanase has been tested according to OECD guidelines, in compliance with GLP. No dermal or eye irritation was observed at any dose site during the studies. The conclusion was that xylanase is neither a skin nor an eye irritant. 

Justification for classification or non-classification

The skin and eye irritation potential of xylanase has been tested according to OECD guidelines and in compliance with GLP. The conclusion was that xylanase did not exert any skin and eye irritation and therfore xylanase should not be classifed.