DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

BCOP Assay OECD 437

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-06-2018 to 20-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 (incl. QA statement)

Test material

Test material information

1

Reference substance name:

DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

EC Number:

212-216-4

EC Name:

DL-α-(aminomethyl)-p-hydroxybenzylic alcohol hydrochloride

Cas Number:

770-05-8

Molecular formula:

C8H11NO2.ClH

IUPAC Name:

4-(2-amino-1-hydroxyethyl)phenol hydrochloride

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder
Batch: D151-1710037
Purity/Composition: 99.8%
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source: Bovine eyes from young cattle were obtained from the
slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands),
where the eyes were excised by a slaughterhouse employee as
soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a
suitable container under cooled conditions.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 µl of a 20% (w/v) suspension of the test item was introduced onto the epithelium of the cornea

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 +- 10 minutes
at 32 +- 1C.

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

no post treatment incubation

Number of animals or in vitro replicates:

3 (three)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. 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
(Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). 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+-1C. The corneas were incubated for the minimum of 1 hour at 32+- 1C.

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 anopacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each corneawas read against a cMEM filled chamber, and the initial opacity reading thus determined wasrecorded. 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.

NEGATIVE CONTROL USED
physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED
The positive control was a 20% (w/v) Imidazole (Merck KGaA, Darmstadt, Germany)
solution prepared in physiological saline.


APPLICATION DOSE AND EXPOSURE TIME
750µL of 20% w/v test article in physiological saline for 240+-10 minutes

TREATMENT METHOD: [closed chamber / open chamber]
closed

POST-INCUBATION PERIOD: NO.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded.

- POST-EXPOSURE INCUBATION:
N/A

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 = [(Io/I) -0.9894]/0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows
and medium, and I 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 item 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 item 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 (Sigma-Aldrich, Germany) 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 (Sigma-Aldrich Chemie GmbH, Germany). 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 +-1C.

Permeability Determinations
After the incubation period, the medium in the posterior compartment of each holder was
removed and placed into a sampling tube labelled according to holder number. 360 l 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 (TECAN Infinite® M200 Pro Plate 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 item was corrected for the mean negative control OD490
before the dilution factor was applied to the reading.
- Others (e.g, pertinent visual observations, histopathology): none

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) :

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

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.
As per the Test guideline

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

ca. 3.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The individual in vitro irritancy scores for the negative controls ranged from -0.9 to 2.7. The
corneas treated with the negative control item were translucent after the 240 minutes of
treatment. All values were within the historical control database. 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 individual positive control in vitro irritancy scores ranged from 144 to 159. The corneas
treated with the positive control were turbid after the 240 minutes of treatment. The mean in
vitro irritancy score of the positive control (20% (w/v) Imidazole) was 153 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: Summary of Opacity, Permeability and In Vitro Scores

Treatment	Mean Opacity	Mean Permeability	Mean IVIS 1,2
negative control	1.2	-0.007	1.1
Positive Control	126	1.776	153
Test Item	3.7	0.000	3.7

1 Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

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

Table 2: Opacity Score

Treatment	Opacity before Treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity^2	Mean Final Opacity
Negative Control	3.5	5.3	1.7		1.2
	4.1	3.3	-0.8
	2.3	5.1	2.8
Positive Control	4.5	123.6	119.1	118	126
	5.4	143.1	137.7	137
	4.1	128.7	124.6	123
Test Item	4.5	10.5	6.0	4.8	3.7
	4.6	8.9	4.3	3.1
	4.5	8.9	4.4	3.2

Calculations are made without rounding off.

1 Final Opacity = Opacity after treatment – Opacity before treatment.

2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

Table 3: Permeability Score Individual Values (Uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative OD
Negative Control	1	-0.008	-0.007	-0.008	-0.008	-0.008	-0.007
	1	-0.011	-0.001	-0.011	-0.008	-0.008
	1	-0.006	-0.006	-0.006	-0.006	-0.006
Positive Control	6	0.409	0.410	0.410	0.410	2.458
	1	1.457	1.468	1.466	1.464	1.464
	1	1.336	1.343	1.368	1.349	1.349
Test Item	1	-0.010	-0.010	-0.009	-0.010	-0.010
	1	-0.004	-0.012	-0.012	-0.009	-0.009
	1	-0.002	-0.002	-0.003	-0.002	-0.002

Table 4: Permeability Score Individual Values (Corrected)

Treatment	Dilution factor	Negative control corrected OD490 1^1	Negative control corrected OD490 2^1	Negative control corrected OD490 3^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive Control	6	0.416	0.417	0.417	0.417	2.501	1.776
	1	1.464	1.475	1.473	1.471	1.471
	1	1.343	1.350	1.375	1.356	1.356
Test Item	1	-0.003	-0.003	-0.002	-0.003	-0.003	0.000
	1	0.003	-0.005	-0.005	-0.002	-0.002
	1	0.005	0.005	0.004	0.005	0.005

1 Calculations are made without rounding off.

OD490 values corrected for the mean final negative control permeability (-0.007).

Table 5: In Vitro Irritancy Score

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy  Score^1
Negative Control	1.7	-0.008	1.6
	-0.8	-0.008	-0.9
	2.8	-0.006	2.7
Positive Control	118	2.501	155
	137	1.471	159
	123	1.356	144
Test Item	4.8	-0.003	4.7
	3.1	-0.002	3.1
	3.2	0.005	3.3

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

2 Positive control and test item are corrected for the negative control.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, since Octopamine hydrochloride induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Octopamine

hydrochloride as 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).

This report describes the potency of chemicals to induce serious eye damage using isolated

bovine corneas.  The eye damage of Octopamine hydrochloride was tested through topical

application for approximately 240 minutes.  

The study procedures described in this report were based on the most recent OECD guideline.

Batch D151-1710037 of Octopamine hydrochloride was a white to off white powder.  The

test item was applied as a 20% (w/v) suspension (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 153 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.  

Octopamine hydrochloride induced ocular irritation through one endpoint (opacity), resulting

in a mean in vitro irritancy score of 3.7 after 4 hours of treatment.

In conclusion, since Octopamine hydrochloride induced an IVIS > 3 ≤ 55, no prediction on

the classification can be made.