Pyridoxine hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Feb 2015 - 10 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Version / remarks:

adopted July 26, 2013

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: In Vitro Techniques in Toxicology Database (INVITTOX) protocol 127. Bovine Opacity and Permeability (BCOP) Assay, 2006

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: The Ocular Toxicity Working Group of the Interagency Coordinating Committee on the Validation of Alternative Methods and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (March 2006)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pyridoxine hydrochloride

EC Number:

200-386-2

EC Name:

Pyridoxine hydrochloride

Cas Number:

58-56-0

Molecular formula:

C8H11NO3.ClH

IUPAC Name:

4,5-bis(hydroxymethyl)-2-methylpyridin-3-ol hydrochloride

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Pyridoxine Hydrochloride (Vitamin B6)
- Substance type: Organic
- Physical state: White powder
- Stability under test conditions: stable
- Storage condition of test material: At room temperature protected from light
- pH: 2.7 at concentration of 5% in water

Test animals / tissue source

Species:

other: in vitro

Test system

Vehicle:

physiological saline

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 μl of a 20% (w/v) suspension

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole

Duration of treatment / exposure:

240 ± 10 minutes

Details on study design:

TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 10 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by incubation for 120 ± 10 minutes in cMEM. Then, corneas were incubated in sodium-fluorescein solution for 90 ± 5 minutes (both incubations at 32 ± 1°C).

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces an IVIS > 55 is defined as a corrosive or severe irritan (UN GHS: category 1);
For a test substance that induces an IVIS >3 and ≥ 55, no prediction on irritant potency can be made (UN GHS: no prediction can be made)

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of three replicates

Value:

94.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with Pyridoxine Hydrochloride (Vitamin B6) showed opacity values ranging from 68 to 106 and permeability values ranging from 0.114 to 0.131. The corneas were turbid after the 240 minutes of treatment with Pyridoxine Hydrochloride (Vitamin B6). A pH effect of the test substance was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed

Any other information on results incl. tables

Summary of opacity, permeability and resulting IVIS

Treatment	Mean opacity ± SD	Mean Permeability ± SD	Mean In vitro Irritation Score ± SD
Negative control	0.0 ± 3.5	0.000 ± 0.004	0.0 ± 3.4
Positive control	87.7 ± 23.1	1.708 ± 0.372	113.3 ± 22.6
Pyridoxine Hydrochloride (Vitamin B6)	92.7 ± 21.4	0.122 ± 0.009	94.5 ± 21.5

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Since Pyridoxine Hydrochloride (Vitamin B6) induced an IVIS ≥ 55 in the Bovine Corneal Opacity and Permeability test performed according to EC/ OECD guidelines and GLP principles, it is concluded that Pyridoxine Hydrochloride (Vitamin B6) should be regarded as severely irritant to the eye (category 1).

Executive summary:

Pyridoxine Hydrochloride (Vitamin B6) was tested in the Bovine Corneal Opacity and Permeability test performed according to EC/ OECD guidelines and GLP principles. The negative control response of the opacity and permeability values was acceptable. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 113 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Pyridoxine Hydrochloride (Vitamin B6) induced serious eye damage through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of 95 after 240 minutes of treatment.

Based on an IVIS of 95, it is concluded that Pyridoxine Hydrochloride (Vitamin B6) induces serious eye damage and is classified category 1 (irreversible effects on the eye).