Reaction mass of 4-(2-methylbutan-2-yl)phenol and 2,4-bis(2-methylbutan-2-yl)phenol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 August 2015 to 01 September 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenol, 1,1-dimethylpropyl derivs.

IUPAC Name:

Phenol, 1,1-dimethylpropyl derivs.

Test material form:

solid

Details on test material:

Identification: Phenol, 1,1-dimethylpropyl derivs.Appearance: Colourless to pale yellow solidBatch OP: C605E003.1Purity/Composition: 100% Unknown or Variable Composition, Complex Reaction Products and Biological Materials (UVCB)Test item storage: At room temperature protected from light container flushed with nitrogenStable under storage conditions until: 31 July 2016 (expiry date)Purity/composition correction factor: No correction factor requiredTest item handling: Use amber glassware or wrap container in aluminum-foil. To liquidify and homogenize: heat the test item until maximum 130°C under nitrogen.Stability at higher temperatures: Yes, maximum temperature: 130°C maximum duration: multiple hours if stored under nitrogen. If heated only minutesChemical name (IUPAC), synonym or trade name: Phenol, 1,1-dimethylpropyl derivs.CAS Number: 1639131-79-5

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 but within 4 hours.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). 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 but within 4 hours after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions obtained using frozen elements which were not in direct contact with the eyes.
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 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 ± 1°C. The corneas were incubated for the minimum of 1 hour at 32 ± 1°C.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

No correction was made for the purity/composition of the test item.Since no workable suspension of Phenol, 1,1-dimethylpropyl derivs. in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.An excessive amount of Phenol, 1,1-dimethylpropyl derivs. applied directly on the corneas in such a way that the cornea was completely covered.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.

Duration of treatment / exposure:

Single exposure

Duration of post- treatment incubation (in vitro):

Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32 ± 1°C.

Number of animals or in vitro replicates:

3 replicates per group (test item, negatice control, positive control).

Details on study design:

Cornea selection and Opacity reading
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be 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. An excessive amount of Phenol, 1,1-dimethylpropyl derivs. applied directly on the corneas in such a way that the cornea was completely covered. 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 item 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.

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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Value:

90

Vehicle controls validity:

not applicable

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 -1.4 to 2.4. The individual positive control in vitro irritancy scores ranged from 145 to 201. The corneas treated with the positive control were turbid after the 240 minutes of treatment.The corneas treated with Phenol, 1,1-dimethylpropyl derivs. showed opacity values ranging from 6.3 to 10.2 and permeability values ranging from 3.550 to 7.204. The corneas were translucent after the 240 minutes of treatment with Phenol, 1,1-dimethylpropyl derivs. And some test item stuck to the cornea. No pH effect of the test item was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 63.5 to 114.4 after 240 minutes of treatment with Phenol, 1,1-dimethylpropyl derivs..

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores

Treatment	Mean Opacity	Mean Permeability	Meanin vitroIrritation Score^1,2
Negative control	0.0	0.000	0.0
Positive control	150.4	1.388	171.3
Phenol, 1,1-dimethylpropyl dervis.	8.8	5.434	90.3

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

Treatment	Opacity before treatment	Opacity after treatment	Final Opacity^1	Negative control corrected Final Opacity^2	Mean Opacity
Negative control	3.6	7.6	4.0	2.5	0.0
	3.2	3.6	0.4	-1.1
	5.1	5.2	0.1	-1.4
		Mean final opacity: 1.5
Positive control	3.9	154.7	150.8	149.3	150.4
	4.5	132.2	127.7	126.2
	3.3	180.6	177.3	175.8
Test item	3.8	15.5	11.7	10.2	8.8
	3.6	11.4	7.8	6.3
	3.5	15	11.5	10.0

Test item = Phenol, 1,1-dimethylpropyl dervis.

¹Final Opacity = Opacity after treatment – Opacity before treatment

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

 

Permeability score individual values (uncorrected)

Treatment	Dilution factor	OD490 1	OD490 2	OD490 3	Average OD	Final OD	Mean final negative control
Negative control	1	0.001	0.001	0.003	0.003	0.003	0.006
	1	0.014	0.004	0.012	0.010	0.010
	1	0.003	0.004	0.005	0.004	0.004
Positive control	6	0.208	0.203	0.213	0.208	1.248
	6	0.218	0.217	0.217	0.217	1.304
	6	0.284	0.288	0.285	0.286	1.714
Test item	6	0.605	0.601	0.586	0.597	3.584
	6	1.213	1.217	1.189	1.206	7.238
	6	0.981	0.912	0.898	0.930	5.582

Test item = Phenol, 1,1-dimethylpropyl dervis.

 

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 OD490Average	Negative control corrected final OD490	Average OD
Negative control	1	-0.005	-0.005	-0.003	-0.004	-0.004	0.000
	1	0.008	-0.002	0.006	0.004	0.004
	1	-0.003	-0.002	-0.001	-0.002	-0.002
Positive control	6	0.202	0.197	0.207	0.202	1.214	1.388
	6	0.212	0.211	0.211	0.212	1.270
	6	0.278	0.282	0.279	0.280	1.680
Test item	6	0.599	0.595	0.580	0.592	3.550	5.434
	6	1.207	1.211	1.183	1.201	7.204
	6	0.975	0.906	0.892	0.925	5.548

Test item = Phenol, 1,1-dimethylpropyl dervis.

¹OD₄₉₀values corrected for the mean final negative control permeability (0.006).

 

In Vitroirritancy score

Treatment	Negative control corrected Final Opacity	Negative control corrected Final OD490	In vitroIrritancy Score^1
Negative control	2.5	-0.004	2.4
	-1.1	0.004	-1.0
	-1.4	-0.002	-1.4
Positive control	149.3	1.214	167.5
	126.5	1.270	145.3
	175.8	1.680	201.0
Test item	10.2	3.550	63.5
	6.3	7.204	114.4
	10.0	5.548	93.2

Test item = Phenol, 1,1-dimethylpropyl dervis.

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

 

Historical control data for the BCOP studies

	Negative control			Positive control
	Opacity	Permeability	In vitroIrritancy Score	In vitroIrritancy Score
Range	-2.3 – 2	-0.056 – 0.050	-2.4 – 2.1	80.3 – 190.3
Mean	-0.17	0.00	-0.23	132.7
SD	0.89	0.01	0.89	23.14
n	97	99	96	102

SD = Standard deviation

n = Number of observations

The above mentioned historical control data of the controls were obtained by collecting all data over the period of August 2012 to August 2015.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Phenol, 1,1-dimethylpropyl derivs. induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 90 after 240 minutes of treatment.Since Phenol, 1,1-dimethylpropyl derivs. induced an IVIS ≥ 55, it is concluded that Phenol, 1,1-dimethylpropyl derivs. induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified Category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.

Executive summary:

Evaluation of the eye hazard potential of Phenol, 1,1-dimethylpropyl derivs. using the Bovine Corneal Opacity and Permeability test (BCOP test).

 

The report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Phenol, 1,1-dimethylpropyl derivs. was tested through topical application for approximately 240 minutes.

 

The study procedures described in the report are in compliance with the following 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 OP: C605E003.1of Phenol, 1,1-dimethylpropyl derivs. was a colourless to pale yellow solid. Since due to physical properties of the test item no workable suspension in physiological saline could be obtained, the test item was used as delivered and an excessive amount was added pure 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 171 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.

 

Phenol, 1,1-dimethylpropyl derivs. induced serious eye damage through both endpoints, resulting in a mean in vitro irritancy score of 90 after 240 minutes of treatment.

 

Since Phenol, 1,1-dimethylpropyl derivs. induced an IVIS ≥ 55, it is concluded that Phenol, 1,1-dimethylpropyl derivs. induces serious eye damage in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report and should be classified Category 1 according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations.