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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 May 2015 to 13 May 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & EU test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
not specified
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
EC Number:
201-044-5
EC Name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Cas Number:
77-62-3
Molecular formula:
C29H40O2
IUPAC Name:
2-{[2-hydroxy-5-methyl-3-(1-methylcyclohexyl)phenyl]methyl}-4-methyl-6-(1-methylcyclohexyl)phenol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: 2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Appearance: White to cream powder
Batch: WWP4J0001
Purity/Composition: 92.2%
Test substance storage: At room temperature
Stable under storage conditions until: 03 September 2018 (retest date)
Test substance handling: No specific handling conditions required
pH (1% in water, indicative range): 6.25 – 6.06 (determined by WIL Research Europe)

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
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, but within maximum 4 hours.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes
Amount / concentration applied:
318.0 to 333.7 mg
Duration of treatment / exposure:
240 minutes of treatment.
Observation period (in vivo):
No observation period required for this study.
Number of animals or in vitro replicates:
3 eyes per group.
Details on study design:
Reference substances
Negative control: A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.
Positive control: 20% (w/v) Imidazole (Merck Schuchardt DHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

Study design
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 (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas, 3 corneas per treatment group, were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont-Ferrand, France) 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.

Cornea selection and Opacity reading
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 (OP-KIT, MC2, Clermont-Ferrand, France). The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

Treatment of corneas and opacity measurements
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. 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (range for 3 eyes 318.0 to 333.7 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. 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 (Eagle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test substance on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance 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.

Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) 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 ± 1°C.

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 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 (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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.

Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
After 240 minutes treatment
Value:
-0.3 - 7
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
After 240 minutes of treatment
Value:
-0.4 - 1.2
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

In vivo

Irritant / corrosive response data:
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints.
Other effects:
None specified

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment

Mean Opacity

Mean Permeability

Mean In vitro Irritation Score1,2

Negative control

0.0

0.000

0.0

Positive control

84.3

3.975

144.0

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

0.0

0.019

0.3

1Calculated using the negative control mean opacity and mean permeability values

2In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value)

 

INDIVIDUAL OPACITY, PERMEABILITY AND IN VITRO SCORES

Opacity score

Eye

Opacity before treatment

Opacity after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Opacity

Negative control

16

1

1

0

-0.3

0.0

17

1

2

1

0.7

18

2

2

0

-0.3

Mean final opacity: 0.3

Positive control

1

0

80

80

79.7

84.3

2

0

74

74

73.7

3

1

101

100

99.7

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

13

2

2

0

-0.3

0.0

14

1

2

1

0.7

15

1

1

0

-0.3

1Final Opacity = Opacity after treatment – Opacity before treatment

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

 

Permeability score individual values (uncorrected)

Eye

Dilution factor

OD490

1

OD490

2

OD490

3

Average OD

Final OD

Mean final negative control

Negative control

16

1

0.012

0.012

0.013

0.012

0.012

0.011

17

1

0.011

0.002

0.003

0.005

0.005

18

1

0.014

0.017

0.015

0.015

0.015

Positive control

1

6

0.833

0.831

0.832

0.832

4.992

 

2

6

0.528

0.532

0.546

0.535

3.212

3

6

0.674

0.649

0.637

0.653

3.920

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]

13

1

0.009

0.009

0.009

0.009

0.009

 

14

1

0.047

0.046

0.050

0.048

0.048

15

1

0.027

0.028

0.043

0.033

0.033

 

Permeability score individual values (corrected)

Eye

Dilution factor

Negative control corrected OD49011

Negative control corrected OD49021

Negative control corrected OD49031

Negative control corrected OC490Average

Negative control corrected final OD490

Average OD

Negative control

16

1

0.001

0.001

0.002

0.001

0.001

0.000

17

1

0.000

-0.009

-0.008

-0.006

-0.006

18

1

0.003

0.006

0.004

0.004

0.004

Positive control

1

6

0.822

0.820

0.821

0.821

4.926

3.975

2

6

0.517

0.521

0.535

0.524

3.146

3

6

0.663

0.638

0.626

0.642

3.854

2,2’-methylenebis[6-(1-methylcycloheyl)-p-cresol]

13

1

-0.002

-0.002

-0.002

-0.002

-0.002

0.019

14

1

0.036

0.035

0.039

0.037

0.037

15

1

0.016

0.017

0.032

0.022

0.022

1OD490values corrected for the mean final negative control permeability (0.011).

 

In Vitro irritancy score

Eye

Negative control corrected Final Opacity

Negative control corrected Final OD490

In vitro Irritancy Score1

Negative control

16

-0.3

0.001

-0.3

17

0.7

-0.06

0.6

18

-0.3

0.004

-0.3

Positive control

1

79.7

4.926

153.6

2

73.7

3.146

120.9

3

99.7

3.854

157.5

2,2’-methylbis[6-(1-methylcyclohexyl)-p-cresol]

13

-0.3

-0.002

-0.4

14

0.7

0.037

1.2

15

-0.3

0.022

0.0

1In vitro irritancy score (IVIS) = opacity value + (15 x OD490value).

 

Historical control data for the BCOP studies

 

Negative control

Positive control

Opacity

Permeability

In vitro Irritancy Score

In vitro Irritancy Score

Range

-3 – 2

-0.056 – 0.050

-3.3 – 2.1

68 – 135

Mean

-0.22

0.00

-0.28

95.22

SD

0.94

0.01

0.95

18.75

N

94

96

93

3

SD = Standard deviation

N = Number of observations

The above mentioned historical control data range of the controls were obtained by collected all data over the period April 2012 to April 2015.

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: OECD GHS
Conclusions:
2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.3 after 240 minutes of treatment.
Executive summary:

Evaluation of the eye hazard potential of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

This report evaluates the potency of chemicals to induce serious eye damage using isolated bovine corneas. The potential eye damaging effects of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was tested through topical application for approximately 240 minutes.

 

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

- Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals; Guideline no. 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”(adopted July 26, 2013).

- European Community (EC). Commission regulation (EC) No. 440/2008, Part B: Methods for the Determination of Toxicity and other health effects, Guideline B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants ". Official Journal of the European Union No. L324; Amended by EC No. 1152/2010 No. L142, 09 December 2010.

 

Batch WWP4J0001 of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was a white to cream powder. Since no workable suspension in physiological saline could be obtained, the test substance was used as delivered and added pure on top of the corneas of three eyes (range for 3 eyes 318.0 to 333.7 mg).

 

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 144 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.3 after 240 minutes of treatment.

Since 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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