Pyridoxine hydrochloride

Endpoint summary

Description of key information

Two studies on skin irritancy are available: (1) An in vitro skin irritation test was conducted according to OECD/EC guidelines and GLP principles. It is concluded that this test is valid and that pyridoxine hydrochloride (Vitamin B6) is not irritating in the in vitro skin irritation test; (2) A 20% solution of pyridoxine chloride was tested on intact and abraded skin of 6 rabbits. No skin irritation was observed. Due to limitations in the study outline it was found to have reliability 4 and is used as supporting evidence. 
Pyridoxine Hydrochloride (Vitamin B6) was tested in the Bovine Corneal Opacity and Permeability test performed according to EC/ OECD guidelines and GLP principles. Since the test substance induced an IVIS ≥ 55, it is concluded that Pyridoxine Hydrochloride (Vitamin B6) should be regarded as severely irritant to the eye. A supporting test is available, in which 6 rabbits were instilled with 0.1 mL of a 15% aqueous solution of the test substance. Mild iriitation was observed which resolved within 48 hours.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 Feb 2015 - 15 feb 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 26 July 2013

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 20 July 2012

Deviations:

no

GLP compliance:

yes

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.7 - 37.2
- Humidity (%): 63-89
- CO2 (%): 5.0 ± 0.5

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 21.0 to 23.0 mg, the skin was moistened with 10 µl Milli-Q water.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

15 ± 0.5 minutes

Number of animals:

3 (number of replicates)

Details on study design:

TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 15-EKIN-006). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 ± 0.5 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Irritation / corrosion parameter:

other: other: tissue viability

Value:

101

Remarks on result:

other:

Remarks:

Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 11%. (migrated information)

Pyridoxine Hydrochloride (Vitamin B6) was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test substance to MTT medium. Because no colour changes were observed it was concluded that Pyridoxine Hydrochloride (Vitamin B6) did not interact with the MTT endpoint.

Interpretation of results:

not classified

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

An in vitro skin irritation test was conducted according to OECD/EC guidelines and GLP principles. It is concluded that this test is valid and that Pyridoxine Hydrochloride (Vitamin B6) is not irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (21.0 to 23.0 mg, in presence of 10 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 11% whereas the test substance showed cell viability of 101%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is non-irritant in the in vitro skin irritation test.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1981

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

No details on substance purity, no Lot/batch number given in the report. Amount applied is not sufficient for classification purposes (0.5 mL of 20% solution). Observation time was 48 hours. No information on test animals given (sex, age, source, clinical signs, body weight measurements).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

yes

Qualifier:

according to guideline

Guideline:

other: Federal Register, 37 (244): 27635-6, December 19, 1972

Principles of method if other than guideline:

Test substance was applied to intact and abraded skin. Exposure was done occlusively instead of semi-occlusively as recommended in the guideline. Observation time was 48 hours instead of 72 hours. Scoring was limited to erythema/ eschar formation and edema formation following the primary index scores listed in the Federal Register 37 (244): 27636, December 19, 1972. No clinical signs or body weight measurements were included. No data on test animals (sex, age, source). The test sites were washed with ethanol.

GLP compliance:

not specified

Species:

rabbit

Strain:

New Zealand White

Type of coverage:

occlusive

Preparation of test site:

other: intact and abraded

Vehicle:

water

Controls:

not required

Amount / concentration applied:

0.5 ml of a 20% solution.

Duration of treatment / exposure:

4 hours

Observation period:

48 hours

Number of animals:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with ethanol
- Time after start of exposure: 4 hours

SCORING SYSTEM: Erythema and eschar formation were scored (scale 0-4) and edema formation was scored (range 0-4), following the primary index scores listed in the Federal Register 37 (244): 27636, December 19, 1972.

Irritation parameter:

erythema score

Basis:

mean

Remarks:

of 6 rabbits

Time point:

other: 24 and 48 hours

Score:

0

Max. score:

4

Remarks on result:

other: Intact and abraded skin

Irritation parameter:

edema score

Basis:

mean

Remarks:

of 6 rabbits

Time point:

other: 24 and 48 hours

Score:

0

Max. score:

4

Remarks on result:

other: Intact and abraded skin

Irritant / corrosive response data:

No skin effects were seen on intact and abraded skin at 24 and 48 hours afer removal of the test substance.

Interpretation of results:

other: data insufficient for classification

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

A 20% solution of pyridoxine chloride was tested on intact and abraded skin of 3 rabbits. No skin irritation was observed.

Executive summary:

A 20% solution of pyridoxine chloride was tested on intact and abraded skin of 3 rabbits. Occlusive exposure of 0.5 ml of the solution did not cause skin irritation. The tested concentration is too low to allow conclusions on irritancy of the pure test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

29 SEP 1981 - 05 OCT 1981

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: No details on test substance purity. amount applied (0.1mL of 15% aquoeus solution) is not sufficient for classification purposes.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

yes

Remarks:

15% solution tested, 6 animals used instead of 3 as recommended

GLP compliance:

no

Species:

rabbit

Details on test animals or tissues and environmental conditions:

IN-LIFE DATES: From: 29 SEP 1981 To: 05 OCT 1981

Vehicle:

water

Remarks:

15% concentration

Controls:

other: untreated eye served as concurrent control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.1 mL
- Concentration (if solution): 15%

Duration of treatment / exposure:

Single instillation

Observation period (in vivo):

14 days

Number of animals or in vitro replicates:

6

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: No

SCORING SYSTEM: According to Draize's method as described in Food and Drug cosmetic Law J. 10: 722 (1955)

Irritation parameter:

cornea opacity score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

conjunctivae score

Remarks:

Redness

Basis:

mean

Remarks:

of 6 animals

Time point:

other: 24 hours

Score:

0.5

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

chemosis score

Basis:

mean

Remarks:

of 6 animals

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

Weak conjunctival irritation of the exposed eyes was seen in all animals 1 hour after instillation and in 3/6 animals after 24 hours (score 1= Some vessels definitely injected). All effects had resolved after 48 hours.

Other effects:

No alteration of the corneas was found macroscopically.

Conclusions:

An in vivo eye irritation test was performed with 6 rabbits. Instillation was done with 0.1 mL of 15% aqueous solution of pyridoxine hydrochloride. Weak conjunctival irritation of the exposed eyes was seen in all animals 1 hour after instillation and in 3/6 animals after 24 hours. All effects had resolved after 48 hours.

Reference 2

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.

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

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

Qualifier:

according to guideline

Guideline:

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

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

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

Species:

other: in vitro

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)

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

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

 

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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two studies are available which address skin irritancy of pyridoxine hydrochloride. Both studies show no indications that the substance has skin irritating properties. In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model), the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm²cultured skin (21.0 to 23.0 mg, in presence of 10 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 11% whereas the test substance showed cell viability of 101%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is non-irritant in the in vitro skin irritation test. Furthermore, a 20% solution of pyridoxine chloride was tested on intact and abraded skin of 3 rabbits. Occlusive exposure of 0.5 ml of the solution did not cause skin irritation. The tested concentration is too low to allow conclusions on irritancy of the pure test substance.

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. Furthermore a supporting study is available. In this in vivo eye irritation test 6 rabbits were instilled with 0.1 mL of a 15% aqueous solution of pyridoxine hydrochloride. Weak conjunctival irritation of the exposed eyes was seen in all animals 1 hour after instillation and in 3/6 animals after 24 hours. All effects had resolved after 48 hours.

Justification for selection of skin irritation / corrosion endpoint:
One study available performed according to OECD/EC guidelines and according to GLP principles.

Justification for selection of eye irritation endpoint:
One study available performed according to OECD/EC guidelines and according to GLP principles.

Effects on eye irritation: highly irritating

Justification for classification or non-classification

Based on the available data, pyridoxine hydrochloride is not classified for irritant properties to the skin, but it is classified for serious effects on the eye (cat.1) according to CLP Regulation (EC) No. 1272/2008.