Xylanase, endo-1,4-

Eye irritation

Administrative data

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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Xylanase, endo-1,4-

EC Number:

232-800-2

EC Name:

Xylanase, endo-1,4-

Cas Number:

9025-57-4

Molecular formula:

Not applicable, see remarks.

IUPAC Name:

endo-1,4-beta-xylanase

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.

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.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Remarks:

Clear brown liquid

Details on test material:

- Substance type: UVCB
- Physical state: liquid
- pH: 4.98
-Specific Gravity: 1.05 g/mL
-Stability (XBU/g): Room Temperature (25°C) stable at 5 hours; % of T=0 was 100%; Refrigerated (4°C) stable at 7 days; % of T=0 was 98.7%; Freezer (-20°C) stable at 90 days; % of T=0 was 96.5%

Test animals / tissue source

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

Test system

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.

Results and discussion

In vivo

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

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).

Applicant's summary and conclusion

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.