Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2017 - 03 October 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 (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

EC Number:

915-318-3

Cas Number:

41199-19-3, 17283-81-7 and 31499-72-6

IUPAC Name:

Reaction mass of 1,2,3,4,4a,5,6,7-octahydro-2,5,5-trimethyl-2-naphthol and 4-(2,6,6-trimethyl-1-cyclohexen-1-yl)butan-2-one and 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)butan-2-one

Test material form:

liquid

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 Vitelco, 's-Hertogenbosch, The Netherlands.
- 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 an transported in physiological saline in a suitable container under cooled conditions.
- 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: 750 µL

NEGATIVE CONTROL: Physiological saline
- Amount applied: 750 µL

POSITIVE CONTROL: Ethanol
- Amount applied: 750 µL

Duration of treatment / exposure:

10 ± 1 minutes (both experiments)

Duration of post- treatment incubation (in vitro):

120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes in sodium-fluorescein for permeability determinations

Number of animals or in vitro replicates:

3 for the negative control, the positive control and the treatment group each for both experiments.

Details on study design:

The experiment was repeated based on a calculation error data. The performed repeat experiment was considered not necessary afterwards. However, the performance of an additional experiment has no impact on the study.

TREATMENT METHOD: The medium from the anterior compartment was removed and 750 µL of either the negative control, positive control or the test item was introduced onto the epithelium of the cornea. The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the control or the test item over the entire cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32.0 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
- POST-EXPOSURE INCUBATION: 120 ±10 minutes in cMEM for opacity measurements and subsequently 90 ±5 minutes 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 (see table 1):
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.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

First experiment; mean of 3 replicates

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 55

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

Second experiment; mean of 3 replicates

Value:

5.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean IVIS: 57

Other effects / acceptance of results:

- 5 out of the 6 individual corneas resulted in an IVIS > 3 ≤ 55
- The corneas treated with the test item showed opacity values between 3.6 and 9.0 in the first experiment and between 0.4 and 2.6 in the second experiment.
- Permeability values were ranging from 0.108 to 0.726 in the first experiment and ranging from 0.070 to 0.670 in the second experiment.
- IVIS scores were 5.2, 9.3 and 20 (n=3) in the first experiment and 3.6, 10 and 2.2 (n=3) in the second experiment.

OTHER EFFECTS:
- No pH effect of the test item was observed on the rinsing medium in both experiments.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range (IVIS ranging from 1.0 to 1.6 in the first experiment and ranging from 0.2 to 0.7 in the second experiment).
- Acceptance criteria met for positive control: yes, results were within historical range (IVIS ranging from 34 to 73 in the first experiment and ranging from 55 to 59 in the second experiment). Corneas were turbid after 10 minutes of treatment in both experiments.

Any other information on results incl. tables

Table 2 Summary of opacity, permeability and in vitro scores in the first experiment

Treatment	Mean opacity^*	Mean permeability^*	Mean IVIS^*#
Negative control	1.2	0.013	1.4
Positive control	18	2.463	55
Test item	6.3	0.326	11

^*Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
^#In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

 

Table 3 Summary of opacity, permeability and in vitro scores in the repeated experiment

Treatment	Mean opacity^*	Mean permeability^*	Mean IVIS^*#
Negative control	0.4	0.002	0.4
Positive control	22	2.299	57
Test item	1.2	0.282	5.4

^*Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
^#In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Table 4 Historical Control Data for the BCOP Studies

	Negative control			Positive control
	Opacity	Permeability	In vitro Irritancy Score	In vitro Irritancy Score
Range	-2.9 – 3.0	-0.016 – 0.042	-2.8 – 3.0	34.7 - 78.2
Mean	0.08	0.01	0.17	56.01
SD	1.04	0.01	1.14	12.51
n	84	77	78	55

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Aug 2014 to Aug 2017.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline 437 and GLP principles, no prediction on the classification of Polyambrol can be made (IVIS > 3 ≤ 55).