1-[3-(Hydroxymethyl)pyridin-2-yl)-4-methyl-2-phenylpiperazine

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 August 2017 - 29 August 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)

Version / remarks:

26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

{2-[(2R)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol; {2-[(2S)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol

EC Number:

700-296-8

Cas Number:

61337-89-1

Molecular formula:

C17-H21-N3-O

IUPAC Name:

{2-[(2R)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol; {2-[(2S)-4-methyl-2-phenylpiperazin-1-yl]pyridin-3-yl}methanol

Test material form:

solid: particulate/powder

Details on test material:

Physical appearance: white powder
Storage conditions: at room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: young cattle, obtained from the slaughterhouse
- Storage, temperature and transport conditions of ocular tissue: eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Subsequently, eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The corneas were incubated for the minimum of 1 hour at 32 ± 1 °C.
- The eyes were checked for unacceptable defects and those exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 321.6 to 387.5 mg (complete coverage of the cornea)

NEGATIVE CONTROL:
- Amount applied: 750 µL

POSITIVE CONTROL:
- Amount applied: 750 µL

Duration of treatment / exposure:

240 +/- 10 minutes

Duration of post- treatment incubation (in vitro):

90 +/- 5 minutes in sodium fluorescein

Number of animals or in vitro replicates:

3

Details on study design:

TREATMENT METHOD:
Two individual experiments were performed. The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (321.6 to 387.5 mg).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
- Number of washing steps after exposure period: After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red.
- POST-EXPOSURE INCUBATION: 90 ±5 minutes 32 ± 1 °C in sodium-fluorescein for permeability determinations.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacity meter (OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microtiter plate reader (TECAN Infinite® M200 Pro Plate Reader, OD490). OD490 values of less than 1.500 were used in the permeability calculation.
- Other: possible pH effects of the test substance on the corneas were recorded.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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

ACCEPTABILITY CRITERIA:
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

Remarks:

mean of 3 replicates

Run / experiment:

Repeated experiment

Value:

65

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 121

Other effects / acceptance of results:

Opacity scores, permeability scores and in vitro irritancy scores are summarized in table 2, 3 and 4 in the section 'any other information on results'.
Since the IVIS were spread over 2 categories (78, 59 and 16), the test was inconclusive and a repeat experiment was performed.

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, in the first experiment, the individual in vitro irritancy scores for the negative controls ranged from -0.6 to 2.8. In the second test, the individual in vitro irritancy scores for the negative controls ranged from 0.1 to 1.4. These results were within the historical data range.
- Acceptance criteria met for positive control: yes, in the first experiment the individual positive control in vitro irritancy scores ranged from 91 to 147 and the corneas treated with the positive control were turbid after the 240 minutes of treatment. In the second experiment, the individual positive control in vitro irritancy scores ranged from 111 to 134 and the corneas treated with the positive control were turbid after the 240 minutes of treatment.

Any other information on results incl. tables

Table 2 Opacity scores

First experiment:

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	3.2	3.0	-0.2			0.7
	3.3	6.1	2.8
	3.8	3.2	-0.6
Positive control	2.7	125.1	122.4	122		104
	4.9	123.8	119.0	118
	2.2	76.1	73.9	73
Test item	3.0	4.4	1.5	0.8		6.7
	1.3	12.4	11.1	10
	3.5	13.2	9.7	9.0

Second experiment

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	2.2	2.0	-0.2			0.4
	2.2	3.5	1.3
	2.7	2.9	0.2
Positive control	3.1	90.0	86.9	86		100
	2.9	116.5	113.6	113
	4.3	105.9	101.5	101
Test item	2.4	5.0	2.6	2.2		0.1
	3.1	3.0	-0.2	-0.6
	4.2	3.2	-1.0	-1.4

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

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

³    Calculations are made without rounding off.

Table 3 Permeability scores (corrected)

First experiment

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	0.277	0.278	0.279	0.278	1.667	1.390
	1	1.308	1.293	1.323	1.308	1.308
	1	1.206	1.183	1.195	1.195	1.195
Test item	1	0.863	0.850	0.864	0.859	5.153	2.964
	1	0.552	0.544	0.533	0.543	3.257
	1	0.489	0.477	0.476	0.481	0.481

Second experiment

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	0.270	0.271	0.276	0.272	1.634	1.391
	1	1.403	1.406	1.402	1.404	1.404
	1	1.129	1.138	1.140	1.136	1.136
Test item	6	0.758	0.761	0.765	0.761	4.568	5.275
	6	0.881	0.883	0.890	0.885	5.308
	6	0.995	0.984	0.995	0.991	5.948

¹  OD490 values corrected for the mean final negative control permeability (0.017).

²    Calculations are made without rounding off.

Table 5 In vitro irritancy score

First experiment

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	-0.2	0.002	-0.2
	2.8	-0.003	2.8
	-0.6	-0.002	-0.6
Positive control	122	1.667	147
	118	1.308	138
	73	1.195	91
Test item	0.8	5.153	78
	10	3.257	59
	9.0	0.481	16

Second experiment

Treatment	Final Opacity^2	Final OD490^2	In vitroIrritancy Score^1
Negative control	-0.2	0.015	0.1
	1.3	0.009	1.4
	0.2	0.027	0.6
Positive control	86	1.634	111
	113	1.404	134
	101	1.136	118
Test item	2.2	4.568	71
	-0.6	5.308	79
	-1.4	5.948	88

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:

Category 1 (irreversible effects on the eye) based on GHS criteria

Remarks:

Category 1 according to Regulation (EC) No. 1272/2008

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, it is concluded that HYDROPIP-NR is classified Category 1 under GHS (IVIS ranging from 71 to 88 after 240 minutes of treatment).

Executive summary:

The eye hazard potential of HYDROPIP-NR was assessed in a Bovine Corneal Opacity and Permeability test (BCOP test), performed according to OECD guideline 437 and GLP principles. Three corneas from young cattle were exposed to 321.6 to 387.5 mg (complete coverage) of the test item, next to a negative control group of physiological saline (n=3) and a positive control of 20% (w/v) imidazole (n=3). Duration of treatment was approximately 240 minutes. Results of the negative and positive controls were within the historical data. Therefore, it was shown that the negative control did not induce irritancy on the corneas, the test conditions were adequate and the test systems functioned properly. Since the IVIS of the test item were spread over 2 categories in the first experiment (78, 59 and 16), the test was inconclusive and a repeat experiment was performed. The results of the repeated experiment showed a mean irritancy score of 65. Based on this result (IVIS > 55), the test item is classified as categry 1 for eye irritation according to GHS criteria and Regulation (EC) No. 1272/2008.