4-(3-methylbut-2-en-1-yl)phenol

Endpoint summary

Description of key information

The test item is considered to be corrosive to skin, since the viability after 3 minutes exposure is lower than 50% and the viability after 1 hour exposure is lower than 15%.

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:

2011-09-28 to 2011-09-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to OECD test guideline 431

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

GLP compliance:

yes (incl. QA statement)

Details on test animals or test system and environmental conditions:

Not applicable - Since this is an in vitro study there is no information on test animals

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL of the undiluted test item was applied to each set of duplicate tissues.

CONTROLS
- Amount(s) applied (volume or weight with unit): 50 µL for positive (Potassium Hydroxyde as 8.0 N KOH) and 50 µL for negative (deionised water) controls were applied to each set of duplicate tissues

Duration of treatment / exposure:

Exposure periods of 3 and 60 minutes

Observation period:

Not applicable

Number of animals:

Not applicable

Details on study design:

CELL CULTURE
- Type: EST-1000™ kits (surface of tissues 0.6 cm^2)
- Source: CellSystems® Biotechnologievertrieb GmbH (53562 St. Katharinen; Germany)
- Storage: Refrigerator at 2 - 8 °C for approximately 1 day
- Acclimation period: At least one hour before stating the assay, tissues were transferred to 6-well plates with maintenance medium which was immediately replaced before the test was started

EXPERIMENTAL PERFORMANCE
Pre-warming of EST-1000™ Tissues:
- At least one hour prior tp dosing the tissues were remove from the refridgerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed medium. Two 24-well plates were prepared as holding plates, , each well containing 1 mL maintenance medium per well. The holding plates were pre-warmed in an incubator (37 +- 1.5 °C, 5 +- 0.5% CO2) until use.

Exposure:
- Duplicate EST-1000™ tissues were exposed to the test item, positive control (potassium hydroxide as 8.0 N KOH) or negative control for each of two different exposure periods: 3 minutes and 1 hour.
After the pre-incubation of the EST-1000™ tissues was completed (1 hour 45 minutes for the 1 hour exposure and 2 hours 10 minutes for the 3 minutes exposure) the medium in each well was replaced by 1.0 mL fresh medium per well. The negative control was added to the surface of duplicate EST-1000™ tissues. Subsequently, the remaining tissues were exposed to the test item and the positive control in the same manner. The 6-well plates were then placed into an incubator (37 +- 1.5 °C, 5 +- 0.5 % CO2).
At the end of the exposure period the tissues were removed from the 6-well plate and gently rinsed using a wash bottle containing PBS to remove any residual test material. Excess PBS was removed by gently shaking the tissue insert and blotting the lower surface with blotting paper. The tissues were placed in the prepared holding plate until all tissues were rinsed.

MTT ASSAY
- After the exposure procedure was completed, the cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) the MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. The inserts were transferred into new 24-well plates. The inserts were immersed in extractant solution by gently pipetting 2 mL of extractant solution (isopropanol) into each insert ensuring that the tissue was completely covered. The 24-well plate was sealed to minimise isopropanol evaporation. The formazan salt was extracted for about 20.5 hours.
After the extraction period the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken, and the insert was discarded. 24-well plates were then placed on a shaker for approx. 15 minutes until the solution was homogeneous in colour.
3 × 200 μL aliquots of the blue formazan solution were transferred from each tissue into a 96-well flat bottom microtiter plate. The optical density (OD) was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) at 570 nm (OD570) without reference filter. The mean values were calculated for each set of 3 wells per tissue insert.

TEST FOR DIRECT MTT REDUCTION BY TEST ITEM:
To check MTT reducing capability of the test item, a solution of MTT in DMEM (1.0 mg/mL) was prepared and each 50 µL of the test item were added to 1 mL MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT.

INTERPRETATION OF RESULTS:
The mean OD value obtained for the duplicate tissues per test item were used to calculate the percent viability relative to the negative control, which was arbitrarily set at 100%.
According to EU CLP Cat.1 and DSD (67/548/EEC):
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

ACCEPTABILITY OF THE ASSAY:
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability met the acceptance criterion if the mean OD570 of the two tissues in both treatment intervals was ≥ 0.8.
The assay met the acceptance criterion if mean relative tissue viability of the positive control was ≤ 30%.

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

8.4

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 min. (migrated information)

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

4.6

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 1 hour. (migrated information)

Irritant / corrosive response data:

The test item is considered to be corrosive to skin, since the viability after 3 minutes exposure is lower than 50% and the viability after 1 hour exposure is lower than 15%.

Table 1: Results after treatment with the test item and the controls

Dose Group	Exposure interval	Absorbance 570 nm Tissue 1*	Absorbance 570 nm Tissue 2*	Mean Absorbance of 2 Tissues	Rel. Absorbance [% of Negative Control]**
Negative Control	3 min	1.722	1.777	1.750	100.0
Positive Control	3 min	0.169	0.189	0.179	10.2
Test item	3 min	0.144	0.151	0.148	8.4
Negative Control	1 hour	1.441	1.786	1.613	100.0
Positive Control	1 hour	0.035	0.032	0.033	2.1
Test item	1 hour	0.070	0.079	0.075	4.6

* Mean of three replicate wells after blank correction

** Relative absorbance [rounded values]: 100 x (absorbance test item) / (absorbance negative control)

The optical evaluation of the MTT-reducing capacity of the test item after one hour incubation with MTT-reagent did not show evidence of a blue colour and thereby was not considered to be an MTT reducer.

Historical Data

- Yes, for 3 minutes treatment positive control (mean viability 11.5% and SD 8.70%) and negative control (mean OD 1.236 and SD 0.264) and for 60 minutes treatment positive control (mean viability 1.95% and SD 2.76%) and negative control (mean OD 1.203 and SD 0.248); Number of studies 86, March 2009 - May 2011.

Interpretation of results:

corrosive

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, it can be stated that in this study and under the reported experimental conditions, the substance was corrosive to skin according to Regulation 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The key study on skin irritation/corrosion is a GLP study according to OECD test guideline 431. The substance was tested to be irritant and corrosive to skin. Skin corrosion is the higher health hazard. Therefore, the study on skin corrosion was used as key study and the study on skin irritation as supporting study.

Justification for selection of skin irritation / corrosion endpoint:
The substance was tested to be irritant and corrosive to skin in GLP studies according to OECD guidelines 439 and 431.

Effects on skin irritation/corrosion: corrosive

Justification for classification or non-classification

This substance is to be labeled as 'H314 Causes severe skin burns and eye damage', the proposed hazard class under Regulation 1272/2008 is 'Skin Corr. 1A'.