Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

GLP compliance:

yes

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

other: Crystalline semi-solid

Details on test material:

Identification: Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate

Specific details on test material used for the study:

Purity/composition correction factor: No correction factor requiredTest item handling: Use amber glassware or wrap container in aluminum-foilStability at higher temperatures: Not stableChemical name (IUPAC), synonym or trade name: Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylateCAS Number: 128840-36-8Molecular formula: C14H24O3Molecular weight: 240.34

Test animals / tissue source

Species:

other: bovine

Strain:

other: Not applicable

Details on test animals or tissues and environmental conditions:

No data, Bovine eyes collected from abattoir straight after slaughter

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: positive and negative control bovine eyes were used

Amount / concentration applied:

750 uL of the control, positive control or test item were apllied to the eppithelium of the cornea.

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

ca. 130 minutes post exposure for opacity, and ca. 220 minutes post exposure for permeability.

Number of animals or in vitro replicates:

N/A

Details on study design:

Bovine corneas were collected immediatly after slaughter and were checked for defects e.g. opacity, scratches, pigmentation and neovascularization whilst being held in the light. Any showing defects were discarded.The isolated corneas were stored in petri dishes with Earle's Minimum Essential Medium (cMEM) containing 1 % (v/v) L-glutamine and 1 % (v/v)Foetal Bovine Serum. The isolated corneas were mounted in a holder (BASF) with the endothelial side against the O-ring of the posterior half of the holder.The corneal holder compartments were filled with cMEM (32 +/- 1 degrees C) and incubated for a minimum of 1 hour at the same tempreture. After incubation the medium was removed and opacity was checked of each cornea using an opacitometer (BASF-OP3.0, Ludwigshafen, Germany). Corneas with an initial opacity reading of more than 7 were discarded, measures were corrected against cMEM only values. Three corneas were selected without bias for each treatment. Medium was added to the posterior compartment at test initiation, with the anterior compartment filled with 750 uL of negative control, positive control (ethanol) or test item. 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 then incubated for 10 minutes, after which solutions were removed and the epithelium washed with MEM (Earle's Minimum Essential Medium only) with Phenol red, and thereafter cMEM. Possible pH effects on the cornea were recorded. The medium in the posterior compartment was then replaced with fresh cMEM. Subsequently the corneas were incubated for 120 +/- 10 minutes at 32  degrees C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.The opacity of a cornea was measured by the diminution of light passing through the cornea (calculation in full report).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.Permeability of the cornea to Na-fluorescein was evaluated after opacity. 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 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. 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  degrees C.After the incubation period the posterior compartments medium was removed and 360 uL from each sample was transferred to a 96-well plate and the optical density was meaured at 490 nm. OD was corrected against a cMEM blank and mean OD was calculated. Mean opacity and permeability was used to estimate the in vitro irritancy score (IVIS) as follows: IVIS = mean opacity + (15 x mean OD490 value)

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro irritation score

Basis:

mean

Time point:

other: 90 miutes

Score:

2.4

Max. score:

6.7

Reversibility:

not specified

Remarks on result:

other: bovine corneas were checked 90 minutes after removal of exposure medium, total exposure to test condition was 10 minutes

Irritant / corrosive response data:

Response data (opactiy and permeability) and historical control data can be seen in Tables 1-6. Mean data and data per replicate are presented for comleteness.The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas (Table 6). The mean in vitro irritancy score of the positive control (Ethanol) was 49 and was within two standard deviations of the current historical positive control mean (Table 1 and Table 5). It was therefore concluded that the test conditions were adequate and that the test system functioned properly.The test item did notinduce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment (Table 1 and 5)

Other effects:

No pH effect of the test item was observed on the rinsing medium.

Any other information on results incl. tables

Table1                 Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity^1	Mean Permeability^1	Mean In vitro Irritation Score^1, 2
Negative control	-0.1	-0.004	-0.2
Positive control (Ethanol)	24.9	1.579	48.6
Test item	1.7	0.053	2.4

 

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 OD₄₉₀value).

Table2              Opacity score

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1		Negative control corrected Final Opacity^2	Mean Final Opacity
Negative control	2.5	1.7	-0.8			-0.1
	2.8	3.0	0.2
	3.5	3.7	0.3
Positive control	3.5	28.0	24.5	24.5		24.9
	3.8	30.9	27.1	27.1
	2.7	25.9	23.2	23.2
Test item	2.9	2.6	-0.2	-0.2		1.7
	2.5	7.5	4.9	4.9
	2.6	2.9	0.3	0.3

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

²   Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control.³

³   In case the mean final opacity of the negative control is below zero, no correction will be made.

⁴  Calculations are made without rounding off.

Table3               Permeability scoreindividual values (uncorrected)

Treatment		Dilution factor	OD490 1	OD490 2		OD490 3		Average OD		Final OD		Mean final negative control
Negative control		1	-0.006		-0.002		-0.002		-0.003		-0.003	-0.004
		1	-0.007		-0.004		-0.004		-0.005		-0.005
		1	-0.008		-0.002		-0.004		-0.005		-0.005
Positive control		6	0.373		0.373		0.374		0.373		2.240
		1	0.629		0.616		0.637		0.627		0.627
		6	0.305		0.302		0.300		0.302		1.814
Test item		1	0.026		0.023		0.023		0.024		0.024
		1	0.120		0.116		0.111		0.116		0.116
		1	0.001		0.010		0.008		0.006		0.006

¹          Calculations are made without rounding off.

Table4               Permeability score individual values (corrected)

Treatment	Dilution factor	Negative control corrected OD4901^1	Negative control corrected OD4902^1	Negative control corrected OD4903^1	Negative control corrected OD490 Average	Negative control corrected final OD490	Average OD
Positive control	6	0.377	0.377	0.378	0.378	2.266	1.579
	6	0.633	0.620	0.641	0.632	0.632
	6	0.309	0.306	0.304	0.307	1.840
Test item	1	0.030	0.027	0.027	0.028	0.028	0.053
	1	0.124	0.120	0.115	0.120	0.120
	1	0.005	0.014	0.012	0.011	0.011

¹          OD₄₉₀values corrected for the mean final negative control permeability (-0.004).

²          Calculations are made without rounding off.

 

Table 5               In Vitro irritancy score

Treatment	Final Opacity^2	Final OD490^2	In vitro Irritancy Score^1
Negative control	-0.8	-0.003	-0.8
	0.2	-0.005	0.1
	0.3	-0.005	0.2
Positive control	24.5	2.266	58.5
	27.1	0.632	36.6
	23.2	1.840	50.8
Test item	-0.2	0.028	0.2
	4.9	0.120	6.7
	0.3	0.011	0.4

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

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

Table6               Historical control data for the BCOP studies

 

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitroIrritancy Score
Range	-2.9 – 3.0	-0.016 – 0.029	-2.8 – 3.0	35.8 – 72.9
Mean	0.25	0.00	0.34	56.58
SD	1.07	0.01	1.18	12.51
n	51	43	45	21

SD = Standard deviation

n = Number of observations

Data over Feb 2015 - May 2016.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

Migrated information No classification Criteria used for interpretation of results: OECD GHS

Conclusions:

Since Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage (ECHA, Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures, Section 3.3, 2015.

Executive summary:

Evaluation of the eye hazard potential of Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylate using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

An excess amount of the test item was applied as it is directly on top of the corneas. The eye damage of the test item was tested through topical application for 10 minutes.

 

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 (Ethanol) was 49 and was within two standard deviations of the current historical positive control mean. One cornea had an opaque off white spot on it, which was assumed not to be an affect of the test item but potentially some undissolved test item.

It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

 

The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 2.4 after 10 minutes of treatment.

 

Since Reaction Mass of 4-tert-butyl-2-hydroxycyclohexyl methacrylate and 5-tert-butyl-2-hydroxycyclohexyl methacrylateinduced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.