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

Endpoint summary

Description of key information

Octopamine hydrochloride was determined non-corrosive in the in vitro skin corrosion test (OECD Guideline 431) under the experimental conditions used; similarly the substance was found to be non-irritant in the in vitro skin irritation test conducted according to OECD Guideline 439.Testing of Octopamine hydrochloride in the BCOP assay induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made. Subsequent testing in a Reconstructed Human EpiOcular™ Model in accordance with OECD guideline 492.  The mean relative tissue viability for Octopamine hydrochloride was below 60% after  6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye. Taking a weight of evidence approach in lieu of in vivo testing, it was determined that Octopamine hydrochloride is a serious eye irritant, based upon the equivocal BCOP result and the positive EpiOcular result.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-06-2018 to 21-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Details on animal used as source of test system:

Mattek corp: Human neonatal foreskin lot number 28831 keratinocyte strain number 00267

Justification for test system used:

Recommended test system in international guidelines (OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm Skin Model (EPI-200, Lot no.: 28831, kit O&P,)
The model consists of normal, human-derived epidermal keratinocytes which have been
cultured to form a multilayered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those
found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate
membranes of 10 mm cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

28.7 to 33.8 mg of the solid test item was added into the 6-well plates on top of the
skin tissues.

Duration of treatment / exposure:

3 minutes and 1 hour

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Two tissues were used for a 3-minute exposure to Octopamine
hydrochloride and two for a 1-hour exposure.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

ca. 94

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

ca. 60

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Octopamine hydrochloride was checked for color interference in aqueous conditions and
possible direct MTT reduction by adding the test item to MTT medium. Because the
solutions did not turn blue / purple nor a blue / purple precipitate was observed it was
concluded that the test item did not interfere with the MTT endpoint.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was
within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance
limit <=2.8) and the laboratory historical control data range. The mean
relative tissue viability following the 1-hour exposure to the positive control was 7.4%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was
<=13%, indicating that the test system functioned properly.

The mean absorption at 570 nm measured after treatment with Octopamine hydrochloride and

controls are presented in Table 1.  

Table 2 shows the mean tissue viability obtained after 3-minute and 1-hour treatments with

Octopamine hydrochloride compared to the negative control tissues.  Skin corrosion is

expressed as the remaining cell viability after exposure to the test item.  The relative mean

tissue viability obtained after the 3-minute and 1-hour treatments with Octopamine

hydrochloride compared to the negative control tissues was 94% and 60% respectively.

Because the mean relative tissue viability for Octopamine hydrochloride was not below 50%

after 3 minutes treatment and not below 15% after 1 hour treatment Octopamine

hydrochloride is considered to be not corrosive.

Table 1

Mean Absorption in the in vitro Skin Corrosion Test with Octopamine hydrochloride

	3 minute application				1 hour application
	A(OD570)	B(OD570)	Mean OD570	SD	A(OD570)	B(OD570)	Mean OD570	SD
Negative Control	1.724	1.762	1.743	0.027	1.814	1.715	1.765	0.070
Test Item	1.745	1.518	1.632	0.160	1.095	1.021	1.058	0.052
Positive Control	0.145	0.103	0.124	0.030	0.120	0.139	0.130	0.014

Table 2: mean Tissue Viability in the in vitro Skin corrosion test with Octopamine hydrochloride

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Octopamine hydrochloride	94	60
Positive control	7.1	7.4

Table 3: Coefficient of Variation between Tissue Replicates

	3 minute	1 hour
Negative Control	2.1	5.5
Octopamine hydrochloride	13	6.8
Positive control	29	14

CV (%) = 100 - [(lowest OD570/highest OD570) x 100%]

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Octopamine hydrochloride is not corrosive in the in vitro skin corrosion test
under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate Octopamine hydrochloride for its ability to induce

skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)).  The

possible corrosive potential of Octopamine hydrochloride was tested through topical

application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD and EC

guidelines.

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

tissue was moistened with 25 µL of Milli-Q water and at least 25 mg of Octopamine

hydrochloride was applied directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7.4% after the 1-hour exposure.

The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was

within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance

limit <=2.8) and the laboratory historical control data range.  In the range of 20 - 100%

viability the Coefficient of Variation between tissue replicates was <=13%, indicating that the

test system functioned properly.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item.  The

relative mean tissue viability obtained after 3-minute and 1-hour treatments with Octopamine

hydrochloride compared to the negative control tissues was 94% and 60%, respectively.

Because the mean relative tissue viability for Octopamine hydrochloride was not below 50%

after the 3-minute treatment and not below 15% after the 1-hour treatment Octopamine

hydrochloride is considered to be not corrosive.

In conclusion, Octopamine hydrochloride is not corrosive in the in vitro skin corrosion test

under the experimental conditions described in this report.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19/JUL/2018 to 19/OCT/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder (determined by Charles River Den Bosch)
Batch: D151-1710037
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 18 EKIN 035). This model is a three-dimensional human epidermis model, which consists of adult humanderived 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. Rationale In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC). Source SkinEthic Laboratories, Lyon, France.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue

Duration of treatment / exposure:

15 ± 0.5 minutes

Duration of post-treatment incubation (if applicable):

42 hour post-incubation period

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Remarks:

Test Item

Value:

> 100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Octopamine hydrochloride was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20152383). Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint. The individual OD570 measurements are presented in Appendix 2. Table 2 shows the mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 106%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 26%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%, indicating that the test system functioned properly.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, Octopamine hydrochloride is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate Octopamine hydrochloride for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SMTM)).  The possible skin irritation potential of the test item was tested through topical application for 15 minutes. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch D151-1710037 of the test item was a white to off white powder.  Skin tissue was moistened with 5 µL of Milli-Q water and at least 10 mg of the test item was applied directly on top of the skin tissue for 15 ± 0.5 minutes.  After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed.  Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test item.  The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test item compared to the negative control tissues was 106%.  Since the mean relative tissue viability for the test item was above 50% after 15 ± 0.5 minutes treatment the test item is considered to be non-irritant. The positive control had a mean cell viability of 26% after 15 ± 0.5 minutes exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.  The standard deviation value of the percentage viability of three tissues treated identically was ≤ 11%, indicating that the test system functioned properly. In conclusion, Octopamine hydrochloride is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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)

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.

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

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.

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.

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.  

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

Epiocular OECD 492

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Jul-2018 to 24-Aug-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Identification: Octopamine hydrochloride
Appearance: White to off white powder (determined by Charles River Den Bosch)
Batch: D151-1710037
Test item storage: At room temperature protected from light
Stable under storage conditions until: 26 October 2019 (retest date)

Species:

human

Details on test animals or tissues and environmental conditions:

Test System
EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27442 Kit B) The EpiOcular tissue construct is a non-keratinized epithelium (0.6 cm2) prepared from normal human keratinocytes (MatTek). It models the cornea epithelium with progressively stratified, but not cornified cells. These cells are not transformed or transfected with genes to induce an extended life span in culture. The “tissue” is prepared in inserts with a porous membrane through which the nutrients pass to the cells. A cell suspension is seeded into the insert in specialized medium. After an initial period of submerged culture, the medium is removed from the top of the tissue so that the epithelial surface is in direct contact with the air. This allows the test material to be directly applied to the epithelial surface in a fashion similar to how the corneal epithelium would be exposed in vivo. Rationale In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro eye irritation tests is the EpiOcular test, which is recommended in international guidelines and scientific publications (e.g. OECD). Source MatTek Corporation, Ashland MA, U.S.A.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 52 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Octopamine hydrochloride (55.8 to 61.5 mg) was applied directly on top of the tissue

Duration of treatment / exposure:

6 hours ± 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

2

Details on study design:

Experimental Design
Test for the Interference of the Test Item with the MTT Endpoint A test item may interfere with the MTT endpoint if it is colored and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed.

Test for Color Interference by the Test Item
Octopamine hydrochloride was checked for possible color interference before the study was started. Some non-colored test items may change into colored items in aqueous conditions and thus stain the tissues during the exposure. To assess the color interference, approximately 50 mg of Octopamine hydrochloride or 50 µL sterile Milli-Q water as a negative control was added to 1.0 mL Milli-Q water. The mixture was incubated for at least 1 hour at 37.0 ± 1.0°C in the dark. Furthermore, approximately 50 mg of Octopamine hydrochloride or 50 µL sterile Milli-Q water as a negative control was added to 2.0 mL isopropanol. The mixture was incubated for 2 - 3 hours at room temperature with gentle shaking. After incubation approximately 1 mL of the mixture was centrifuged for 30 seconds at 16000 g. At the end of the exposure time, the absorbance of the solutions was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. If after subtraction of the negative control, the OD for the test item solution is >0.08, the test item is considered as possibly interacting with the MTT measurement. 4.6.1.2. Test for Reduction of MTT by the Test Item Octopamine hydrochloride was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 50 mg of Octopamine hydrochloride was added to 1 mL MTT solution (1 mg/mL MTT in phosphate buffered saline). The mixture was incubated for approximately 3 hours at 37.0 ± 1.0°C in the dark. A negative control, 50 µL sterile Milli-Q water was tested concurrently.
If the MTT solution color turned blue / purple or if a blue / purple precipitate was observed the test item interacts with MTT. Only test items which bind to the tissue after rinsing can interact with MTT in the main assay.

Test System Set Up: On the day of receipt the tissues were equilibrated (in its 24-well shipping container) to room temperature. Subsequently, tissues were transferred to 6-well plates and incubated for 20 ± 4 hours at 37°C in 1.0 mL fresh pre-warmed Assay Medium. Assay Medium was supplied by MatTek Corporation, Ashland, USA. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation. MTT medium MTT concentrate (5 mg/mL) diluted (1:5) with MTT diluent. Environmental conditions All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 52 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 - 37.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Test Item Preparation: No correction was made for the purity/composition of the test item. The solid test item (55.8 to 61.5 mg) was applied directly on top of the skin tissue. Any residual volumes were discarded.

Application/Treatment of the Test Item: The test was performed on a total of 2 tissues per test item together with a negative control and positive control. Before the assay was started the entire tissues were pre-wetted with 20 μL of Ca2+Mg2+-FreeDPBS. The tissues were incubated at standard culture conditions for 30 ± 2 minutes. Two tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues with 50 µL Methyl Acetate (positive control) respectively.
At least 50 mg solid was added into the 6-well plates on top of the tissues. After the exposure period with Octopamine hydrochloride (6 hours ± 15 minutes at 37.0 ± 1.0°C), the tissues were thoroughly rinsed with Ca2+Mg2+-free D-PBS (brought to room temperature) to remove residual test item. After rinsing the cell culture inserts were each dried carefully and immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for a 25 ± 2 minute immersion incubation at room temperature (Post-Soak). After the Post-Soak period cell culture inserts were each dried carefully and transferred to the 6-well plate containing 1.0 mL of warm Assay Medium and were incubated for 18 hours ± 15 minutes at 37°C. 4.6.5. Cell Viability Measurement After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 180 ± 10 minutes at 37°C. After incubation with MTT-medium the tissues were placed on blotting paper to dry the tissues and then transferred to a pre-labeled 6-well plate containing 2 mL isopropanol in each well so that no isopropanol is flowing into the insert. Formazan was extracted with 2 mL isopropanol refrigerated overnight in the dark. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

Irritation parameter:

in vitro irritation score

Remarks:

% MEAN CELL TISSUE VIABILITY

Run / experiment:

TEST ITEM

Value:

ca. 3.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Interference of the Test Item with the MTT Endpoint
Octopamine hydrochloride was checked for possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that Octopamine hydrochloride did not interact with the MTT endpoint. Octopamine hydrochloride was checked for color interference in aqueous conditions. Addition of the test item to Milli-Q and isopropanol resulted after subtraction of the blank in an OD of 0.0013 and 0.0008, respectively. Therefore it was concluded that the test item did not induce color interference.
Main Assay: The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with Octopamine hydrochloride compared to the negative control tissues was 3.3%. Since the mean relative tissue viability for Octopamine hydrochloride was below 60% it is considered to be potentially irritant or corrosive to the eye. The positive control had a mean cell viability after 6 hours ± 15 minutes exposure of 15%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 2%, indicating that the test system functioned properly.

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In conclusion, Octopamine hydrochloride is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report. The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Executive summary:

The objective of this study was to evaluate the eye hazard potential of Octopamine hydrochloride.  For this purpose Octopamine hydrochloride was topically applied on the Reconstructed Human EpiOcular™ Model. The possible eye hazard potential of Octopamine hydrochloride was tested through topical application for 6 hours. 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.  Octopamine hydrochloride (55.8 to 61.5 mg) was applied directly on top of the tissue for  6 hours ± 15 minutes.   After exposure the cornea epithelial construct was thoroughly rinsed to remove the test item and transferred to fresh medium for an immersion incubation.  Afterwards, the tissues were transferred to fresh medium and incubated for 18 hours at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 15% after 6 hours ± 15 minutes exposure.  The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range.  The difference between the percentage of viability of two tissues treated identically was less than 2%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test item.  The relative mean tissue viability obtained after 6 hours ± 15 minutes treatment with Octopamine hydrochloride compared to the negative control tissues was 3.3%.   Since the mean relative tissue viability for Octopamine hydrochloride was below 60% after  6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye.   In conclusion, Octopamine hydrochloride is potentially irritant or corrosive in the EpiOcular™ test under the experimental conditions described in this report.  The test item is identified as potentially requiring classification and labelling according to UN GHS (Category 2 or Category 1).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Octopamine hydrochloride was determined non-corrosive in the in vitro skin corrosion test (OECD Guideline 431) under the experimental conditions used; similarly the substance was found to be non-irritant in the in vitro skin irritation test conducted according to OECD Guideline 439.

According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Octopamine hydrochloride does not have to be classified and has no obligatory labelling requirement for dermal irritaiton or corrosion.

Testing of Octopamine hydrochloride in the BCOP assay induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made. Subsequent testing in a Reconstructed Human EpiOcular™ Model in accordance with OECD guideline 492.  The mean relative tissue viability for Octopamine hydrochloride was below 60% after  6 hours ± 15 minutes treatment it is considered to be potentially irritant or corrosive to the eye. Taking a weight of evidence approach in lieu of in vivo testing, it was determined that Octopamine hydrochloride is a serious eye irritant, based upon the equivocal BCOP result and the positive EpiOcular result. According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), Octopamine hydrochloride should be classified as a serious eye irritant and has an obligatory labelling requirement for eye irritation category 2.