Thiamine hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 June 2017 to 30 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Thiamine hydrochloride

EC Number:

200-641-8

EC Name:

Thiamine hydrochloride

Cas Number:

67-03-8

Molecular formula:

C12H17N4OS.ClH.Cl

IUPAC Name:

thiamine hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White powder
- Test item storage: At room temperature protected from light and humidity

Specific details on test material used for the study:

- No correction was made for the purity/composition of the test material.
- A solubility test in physiological saline was performed based on visual assessment.
- A 20% (w/v) solution of the test material was prepared in physiological saline.
- Any residual volumes were discarded.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse, where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter.
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularisation by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: None reported

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 750 µL
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied: 750 µL

Duration of treatment / exposure:

240 ± 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- The eyes were checked for unacceptable defects; those exhibiting defects were discarded.
- The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1 % (v/v) L-glutamine and 1 % (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 ± 1 °C. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
- After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used.

NUMBER OF REPLICATES
- Three corneas were selected at random for each treatment group.

VEHICLE CONTROL USED: physiological saline

POSITIVE CONTROL USED: 20% (w/v) Imidazole solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 240 ± 10 minutes at 32 ± 1 °C.

TREATMENT METHOD
- The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or 20 % (w/v) solution of the test material was introduced onto the epithelium of the cornea.
- The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- After the incubation the solutions and the test material were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies).
- Post-exposure incubation: Yes, with sodium fluorescein.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed. The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter. The opacity value (measured with the device OP-KIT) was calculated according to: Opacity = [(I0 / I) - 0.9894] / 0.0251
- With I0 being the empirically determined illuminance through a cornea holder but with windows and medium and I being the measured illuminance through a holder with cornea. The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test material or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test material or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: Following the final opacity measurement, permeability of the cornea to Na-fluorescein was evaluated. The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 mL of 5 mg Na-fluorescein/mL cMEM solution. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32 ± 1 °C. After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader. Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test material was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
- Others: Possible pH effects of the test material on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
- 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)
Additionally the opacity and permeability values were evaluated independently to determine whether the test material induced irritation through only one of the two endpoints.

DECISION CRITERIA
- The IVIS cut-off values for identifying the test materials as inducing serious eye damage (UN GHS Category 1) and test materials not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are: In vitro score range: ≤ 3 = UN GHS No Category; > 3 but ≤ 55 = No prediction can be made; and >55 = UN GHS Category 1

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if:
- The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

17

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

TEST MATERIAL
- The corneas treated with the test material showed opacity values ranging from 6.2 to 24 and permeability values ranging from 0.016 to 0.071. The corneas were translucent after the 240 minutes of treatment with the test material.
- A pH effect of the test material was observed on the rinsing medium, the corneas were rinsed until no colour change of the medium was observed. Hence, the in vitro irritancy scores ranged from 6.4 to 25 after 240 minutes of treatment with the test material.
- The test material induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 17 after 240 minutes of treatment.

CONTROL GROUPS
- The individual in vitro irritancy scores for the negative controls ranged from -0.1 to 2.3.
- The individual positive control in vitro irritancy scores ranged from 124 to 150. The corneas treated with the positive control were turbid after the 240 minutes of treatment.
-The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 134 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Table 1: Opacity, Permeability and In Vitro Scores

Treatment		Final Opacity	Final OD490	IVIS
Negative control	1	2.1	0.008	2.3
	2	-0.1	0.015	0.1
	3	-0.1	0.001	-0.1
	Mean	0.6	0.008	0.7
Positive control	1	91	2.167	124
	2	102	1.773	129
	3	102	3.183	150
	Mean	98	2.375	134
Test material	1	6.2	0.017	6.4
	2	24	0.071	25
	3	18	0.016	18
	Mean	16	0.034	17

Applicant's summary and conclusion

Interpretation of results:

other: No prediction on the classification can be made

Conclusions:

Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.

Executive summary:

The potential of the test material to cause irritation to the eye was investigated in accordance with the standardised guideline OECD 437, under GLP conditions.

The eye hazard potential of the test material was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test).

The eye damage of the test material was tested through topical application for approximately 240 minutes. The test material was applied as a 20% (w/v) solution (750 μL) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 134 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Thiamine hydrochloride induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 17 after 4 hours of treatment.

Under the conditions of this study, the test material induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made.