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

Endpoint summary

Description of key information

Skin Irritation/Corrosion – In Vitro

Key value determined in a GLP accredited laboratory study using a human skin model, performed in accordance with OECD Guideline 431 and EU Method B.40 BIS.

Eye Irritation - In Vitro

Key value determined in a GLP accredited laboratory study using BCOP test performed in accordance with OECD Guideline 437 and EU Method B.47.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2015 to 25 September 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.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: (EC) No. 440/2008, Guideline B.40 BIS: "In Vitro Skin Corrosion: Human Skin Model Test".

Deviations:

no

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Amount/concentration applied:

EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and J).
The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts.

Species:

other: EpiDerm Skin Model

Strain:

other: Not applicable

Details on test animals or test system and environmental conditions:

Test system: EpiDerm Skin Model (EPI-200, Lot no.: 22676 kit K and J). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. Rationale: Recommended test system in international guidelines (OECD and EC). Source: MatTek Corporation, Ashland MA, U.S.A.Cell culture Tissues On the day of receipt the tissues were kept on agarose gel and stored in the refrigerator. On the next day, at least one hour before starting the assay the tissues were transferred to 6-well plates with 0.9 ml DMEM medium. Freeze-killed tissues (EPI-200, Lot no.: 22268 kit H and 22299 kit O) Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium. Further use of killed tissues was similar to living tissues. DMEM (Dulbecco’s Modified Eagle’s Medium) Supplemented DMEM medium, serum-free supplied by MatTek Corporation. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) medium MTT concentrate (5 mg/ml) diluted (1:5) with MTT diluent (supplemented DMEM). Both supplied by MatTek Corporation. Environmental conditions All incubations, with the exception of the test item incubation of 3 minutes at room temperature, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 58 - 90%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.3 - 37.3 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Type of coverage:

open

Preparation of test site:

other: Not applicable

Vehicle:

water

Controls:

yes

Amount / concentration applied:

approximately 25 mg of the test item

Duration of treatment / exposure:

3 minutes & 1 hour

Observation period:

No observation period required for the study design

Number of animals:

4 tissues

Details on study design:

Test for the interference of the test item with the MTT endpoint A test item may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test item is present on the tissues when the MTT viability test is performed. Test for colour interference by the test item The test item was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, approximately 25 mg of the test item or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed. In case the test item induces colour interference in aqueous conditions, in addition to the normal procedure, two tissues must be treated with test item for 3 minutes and two tissues for 1-hour. Instead of MTT solution these tissues will be incubated with DMEM medium. Test for reduction of MTT by the test item Phenol, 1,1-dimethylpropyl derivs. was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, approximately 25 mg of the test item was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C. A negative control, sterile Milli-Q water was tested concurrently.In case the test item reacts with the MTT medium in addition to the normal 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed non treated tissues must be used for the cytotoxicity evaluation with MTT. At the end of the exposure time it was checked if a blue / purple colour change was observed. Application/Treatment of the test item The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay is started the tissues were transferred to 6-well plates containing 0.9 ml DMEM medium per well. The level of the DMEM medium is just beneath the tissue (see fig 1). The plates were incubated for approximately 1.5 hour at 37.0 ± 1.0°C. The medium was replaced with fresh DMEM medium just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to Phenol, 1,1-dimethylpropyl derivs. and two for a 1-hour exposure. The skin was moistened with 25 μl Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and an excessive amount of the solid test item was added into the 6-well plates on top of the skin tissues. The remaining tissues were treated with 50 μl Milli-Q water (negative control) and with 50 μl 8N KOH (positive control), respectively. In addition for the 3 minute and 1 hour exposure two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT. After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. Rinsed tissues were kept in 24 well plates on 300 μl DMEM medium until 6 tissues (= one application time) were dosed and rinsed.Cell viability measurement The DMEM medium was replaced by 300 μl MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 ml isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as percentage of the mean of the negative control tissues. Skin corrosion potential of the test item was classified according to remaining cell viability following exposure of the test item with either of the two exposure times.

Irritation / corrosion parameter:

other: other: mean tissue viability

Value:

52

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minute. Reversibility: no data. (migrated information)

Irritation / corrosion parameter:

other: other: mean tissue viability

Value:

11

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour. Reversibility: no data. (migrated information)

Irritant / corrosive response data:

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 52% and 11% respectively. Because the mean relative tissue viability for the test item was below 15% after 1 hour treatment it is considered to be corrosive.

Other effects:

Phenol, 1,1-dimethylpropyl derivs. was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by the test item was 0.29% and 23% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The mean relative tissue viability following 3-minute exposure to the positive control was 12%. The maximum inter-tissue variability in viability between two tissues treated identically was less than 16% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 8% in the range of 20 – 100% viability. In the range of <20% viability, the maximum inter-tissue variability in viability between two tissues treated identically was less than 49% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 33%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.

Mean absorption in the in vitro skin corrosion test with Phenol, 1,1-dimethylpropyl dervis.

	3-minute application					1-hour application
	A (OD570)	B (OD570)	Mean (OD570)		SD	A (OD570)	B (OD570)	Mean (OD570)		SD
Negative control	2.108	2.211	2.160	±	0.073	2.144	2.220	2.182	±	0.053
Phenol, 1,1-dimethylpropyl dervis^. (1)	1.223	1.039	1.131	±	0.130	0.164	0.319	0.241	±	0.110
Positive control	0.296	0.206	0.251	±	0.064	0.174	0.217	0.195	±	0.030

SD = Standard deviation

Duplicate exposure are indicated by A and B

(1) The values are corrected for the non-specific MTT reaction.

In this table the values are corrected for background absorption (0.0423). Isopropanol was used to measure the background absorption.

 

Mean tissue viability in the in vitro skin corrosion test with Phenol, 1,1-dimethylpropyl dervis.

	3-minute application viability (percentage of control)	1-hour application viability (percentage of control)
Negative control	100	100
Phenol, 1,1-dimethylpropyl dervis.	52	11
Positive control	12	9

 

INDIVIDUAL OD MEASUREMENTS AT 570NM

	3-minute application (OD^570)		1-hour application (OD570)
	A	B	A	B
Negative control OD570measurement 1 OD570measurement 2 OD570measurement 3	2.1091 2.1567 2.1865	2.2521 2.2835 2.2246	2.1831 2.2167 2.1602	2.2204 2.2843 2.2822
Phenol, 1,1-dimethypropyl dervis. OD570measurement 1 OD570measurement 2 OD570measurement 3	1.3610 1.2910 1.1627	1.1178 1.1074 1.0486	0.7161 0.7172 0.7151	0.8907 0.8467 0.8775
Negative control treated freeze killed tissue OD570measurement 1 OD570measurement 2 OD570measurement 3	0.1722 0.1670 0.1689	0.1747 0.1776 0.1724	0.1656 0.1697 0.1696	0.1631 0.1619 0.1599
Positive control OD570measurement 1 OD570measurement 2 OD570measurement 3	0.3182 0.3452 0.3513	0.2518 0.2487 0.2446	0.2180 0.2157 0.2153	0.2575 0.2559 0.2635

OD = Optical density

Duplicate exposure are indicated by A and B.

 

HISTORICAL CONTROL DATA FOR IN VITRO SKIN CORROSION STUDIES

	Negative control		Positive control		Positive control
	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (OD570)	1-hour treatment (OD570)	3-minute treatment (% viability)	1-hour treatment (% viability)
Range	1.076 – 2.167	1.361 – 2.203	0.017 – 0.29	0.063 – 0.226	6 – 16	4 – 12
Mean	1.78	1.78	0.16	0.12	10.6	7.0
SD	0.25	0.20	0.05	0.04	2.9	2.1
N	43	47	44	42	22	22

SD = Standard deviation

N = Number of observations

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

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Phenol, 1,1-dimethylpropyl derivs. is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Executive summary:

In vitro skin corrosion test with Phenol, 1,1-dimethylpropyl derivs. using a human skin model.

This report describes the ability of Phenol, 1,1-dimethylpropyl derivs. to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of the test item was tested through topical application for 3 minutes and 1 hour.

 

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

 

Batch OP: C605E003.1 of the test item was a colourless to pale yellow solid. Skin tissue was moistened with 25 μl of Milli-Q water and an excess amount of the test item was applied directly on top of the skin tissue.

 

The positive control had a mean relative tissue viability of 12% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

The test item did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).

In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each time point. The non-specific reduction of MTT by the test item was 0.29% and 23% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

 

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 52% and 11%, respectively. Because the mean relative tissue viability for the test item was below 15% after the 1-hour treatment it is considered to be corrosive.

 

Finally, it is concluded that this test is valid and that Phenol, 1,1-dimethylpropyl derivs. is corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

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

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

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)

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.

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.

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..

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Justification for classification or non-classification

Skin Irritation/Corrosion – In Vitro

It is concluded that this test is valid and that Phenol, 1,1-dimethylpropyl derivs. is corrosive in the in vitro skin corrosion test under the experimental conditions described in the report.

Eye Irritation - In Vitro

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.