2-Cyclopentene-1-acetic acid, ethyl ester

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2011-12-14 to 2012-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study reliable without restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2010-07-22

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

2009-08-24

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation (Altern Lab Anim. 2007 Dec; 35 (6):559-601)

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2009-03-30

Test material

Test material information

Constituent 1

Reference substance name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

EC Number:

605-150-6

Cas Number:

15848-49-4

Molecular formula:

C9H14O2

IUPAC Name:

ethyl 2-(cyclopent-2-en-1-yl)acetate

Test material form:

other: liquid

Details on test material:

- Physical state: liquid
- Molecular formula: C9H14O2
- Molecular weight: 154.21 g/mol

Test animals

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.

Test system

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 μL of the undiluted test item were applied to each of triplicate tissues (Experiment 1 and 2)

Duration of treatment / exposure:

15 ± 1 minutes

Observation period:

not applicable

Number of animals:

not applicable

Details on study design:

NOTE: Due to a borderline result (Experiment 1), a second (Experiment 2), confirming experiment was performed.

CELL CULTURE:
EpiSkin™ Kits (Experiment 1: Lot No.: 11-EKIN-046; Experiment: 12-EKIN-006) were purchased from SkinEthic Laboratories (06000 Nice, France). The EpiSkin™ tissue 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 EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts. EpiSkin™ tissues were shipped with ice packs on medium-supplemented agarose gels in a 12-well plate and reached Harlan CCR on December 13, 2011 (Experiment 1) or on February 07, 2012 (Experiment 2), respectively. On day of experiments EpiSkin™ tissues were transferred to 12-well plates with maintenance medium.

TEST FOR DIRECT MTT REDUCTION
For correct interpretation of results it was necessary to assess the ability of the test item to directly reduce MTT. To test for this ability 10 µL of the test item were added to 1 mL of MTT solution and the mixture was incubated in the dark at room temperature for 60 minutes. Untreated MTT medium was used as control. If the MTTsolution colour turned blue/ purple, the test item was presumed to have reduced the MTT. A colour change could not be observed.

TREATMENT:
- Prewarming of EpiSkin™ tissues: after nearly 24 hours incubation of the EpiSkin™ tissues in Experiment 1 and after about six hours incubation in Experiment 2, they were treated with the test item. Under sterile conditions using sterile forceps, the inserts were transferred into 12-well plates containing the pre-warmed (37 ± 1.5 °C) maintenance medium.
- The negative control (deionised water (Lot nos. 23.11.11 (Experiment 1) and 260112 (Experiment 2); Volume 10 µL) and positive control (5% SLS (Sodium lauryl sulphate, Fluka, Sigma-Aldrich 89555 Steinheim) solution in deionised water, prepared freshly prior to the performance of the experiment; Volume 10 µL), and the test item were added into the insert atop the concerning EpiSkin™ triplicate tissues each.
- The 12-well plates were placed into the incubator for 15 ± 1 min at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- After the end of the treatment interval the inserts were removed immediately from the 12-well plate. Using a wash bottle the tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.
- The inserts were placed in the plates with 2 mL maintenance medium. The tissues were incubated for about 42 hours at 37 ± 1.5 °C, 5 ± 0.5 % CO2.
- After the further incubation for about 42 hours the tissues were treated with the MTT solution for 3 hours followed by approximately 72 hours (Experiment 1) or about 69 hours (Experiment 2) extraction of the colorant cells (cell viability test).
- The amount of extracted colorant was determined photometrically at 570 ± 1 nm.

CELL VIABILITY TEST:
Cell viability is measured by dehydrogenase conversion of MTT [(3-4,5-dimethyl thiazole 2-yl) 2,5-diphenyl-tetrazoliumbromide], in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues (Faller, C., Bracher, M., Dami, N., Roguet, R., 2002. Predictive ability of reconstructed human epidermis equivalents for assessment of skin irritation of cosmetics. Toxicology in vitro 16 (5), 557-552).
The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential (see OECD TG 439) and is used for the purpose of classification as irritating or non-irritating according to chemicals law (EU CLP, UN GHS).
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to plates filled with 2 mL assay medium containing 0.3 mg/mL MTT per well.
After a 3 hour incubation period (37 ± 1.5°C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and the wells were rinsed three times with PBS. Tissue samples were cut out of the inserts with a biopsy punch and transferred into plastic vials. The tissue samples were immersed into extractant solution by gently pipetting 0.5 mL extractant solution (isopropanol/2 N HCl 49:1 (v/v)) into each vial. The tissue samples were completely covered by isopropanol. The vials were sealed to inhibit isopropanol evaporation. The formazan salt was extracted for approximately 72 hours in Experiment 1 and for about 69 hours in Experiment 2, each time in the refrigerator.
Per each tissue sample 2 X 200 µL aliquots of the formazan blue solution were transferred into a 96-well flat bottom microtiter plate. OD was read in a microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany) with 570 ± 1 nm filter. Mean values were calculated from the 2 wells per tissue sample.

EVALUATION OF RESULTS:
The mean OD of the three negative control tissues was calculated. This value corresponds to 100% tissue viability in the current test. For each individual tissue treated with the test item or the positive control the individual relative tissue viability is calculated according to the following formula:
Relative viability (%) = [OD test item/ OD mean of negative control] X 100
For the test item and the positive control the mean relative viability ± standard deviation of the three individual tissues are calculated and used for classification according to the following prediction model:
For the current test, an irritation potential of a test item according to EU classification R38 (accoding to directive 67/548/EEC), H315 (according to regulation (EC) 1272/2008) is recommended if the mean relative tissue viability of three individual tissues is reduced below 50% of the negative control.

ACCEPTABILITY OF THE ASSAY:
The absolute OD 570 nm of the negative control tissues in the MTT test is an indicator of tissue viability obtained after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is meeting the acceptance criterion if the mean OD of the three tissues is ≥ 0.6 till ≤ 1.5.
The standard deviations in between tissues of the same treatment group should be ≤ 18%.
An assay is meeting the acceptance criterion if mean relative tissue viability of the positive control is ≤ 40%.
The acceptance limit of IC50 for the respective EpiSkin™ lot should be between 1.0 and 3.0 mg/mL after 18 hours treatment with SLS.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: relative viability(%)

Value:

49.5

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 15 min incubation. Reversibility: no data. Remarks: Result from Experiment 1. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: relative viability (%)

Value:

5.7

Remarks on result:

other:

Remarks:

Basis: mean. Time point: after 15 min incubation. Reversibility: no data. Remarks: Result from Experiment 2. (migrated information)

In vivo

Irritant / corrosive response data:

After exposure of the test item the mean relative absorbance value decreased to 49.5% in the first experiment. This value is tightly below the threshold for irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential. Due to this borderline result, a second, confirming experiment was performed.
After treatment with the test item in the second experiment the mean relative absorbance value decreased to 5.7%. This value is clearly below the threshold for
irritancy of ≤ 50%. Therefore, the test item is considered to possess an irritant potential.

Any other information on results incl. tables

HISTORICAL DATA

Positive Control		Negative Control
Number of Studies	122	Number of Studies	122
Period	July 2007 – May 2011	Period	July 2007 – May 2011
Mean Viability	17.8%	Mean OD	1.051
Standard Deviation	10.7%	Standard Deviation	0.185
Range of Viabilities	0.7% - 39.7%	Range of ODs	0.59 – 1.68

Results after treatment with the test item (Experiment 1)

Dose group	Treatment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [% of Negative Control]**
Negative Control	15 min	0.843	0.766	0.702	0.770	100.0
Positive Control	15 min	0.127	0.382	0.266	0.259	33.6
Test item	15 min	0.401	0.336	0.406	0.381	49.5

* Mean of two replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance_testitem)/(mean absorbance negative control)

Results after treatment with the test item (Experiment 2)

Dose group	Treatment Interval	Absor-bance 570 nm Tissue 1*	Absor-bance 570 nm Tissue 2*	Absor-bance 570 nm Tissue 3*	Mean Absor-bance of 3 Tissues	Rel. Absor-bance [% of Negative Control]**
Negative Control	15 min	1.233	1.205	1.018	1.152	100.0
Positive Control	15 min	0.245	0.298	0.216	0.253	22.0
Test item	15 min	0.044	0.053	0.100	0.066	5.7

* Mean of two replicate wells after blank correction

** relative absorbance per treatment group [rounded values]: 100 x (mean absorbance_testitem)/(mean absorbance negative control)

- After treatment with the negative control the absorbance values were well within the required acceptability criterion of mean OD ≥ 0.6 till ≤ 1.5 for the 15 minutes treatment interval in both experiments, thus showing the quality of the tissues.

- Treatment with the positive control induced a decrease in the relative absorbance compared with the negative control to 33.6% in the 1st experiment and to 22.0% in the 2nd experiment, thus ensuring the validity of the test system.

- The standard deviations between the % variabilities of the test item, the positive and negative controls were below 17% in the 1st experiment and below 11% in the 2nd experiment (threshold of the "OECD TG 439 Guideline for the Testing of Chemicals 439: In vitro Skin Irritation: Reconstructed Human Epidermis Test Method”: 18%), thus ensuring the validity of the study.

- Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue colour in both experiments.

Applicant's summary and conclusion

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is irritant to the skin.
According to the EC-Commission directive 67/548/EEC and its subsequent amendments, the test substance is irritant to the skin.
According to the EC-Regulation 1272/2008 and subsequent regulations, the test item is classified as Category 2, H315.