Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 - 16 Nov 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Function Test (Altern Lab Anim. 2007 Dec.; 35 (6): 559-601)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Protocol for: IN VITRO EpiDerm™ SKIN IRRITATION TEST (EPI-200-SIT), Rev. 3/23/2009, MatTek Corporation, Ashland, USA

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane

EC Number:

306-085-3

EC Name:

Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane

Cas Number:

95912-89-3

Molecular formula:

not applicable, UVCB

IUPAC Name:

Fatty acids, C8-10 (even numbered), mixed esters with adipic acid and trimethylolpropane

Details on test material:

- Name of test material (as cited in study report): Fatty acids, C8-10, mixed esters with adipic acid and trimethylolpropane
- Physical state: yellowish brown liquid
- Analytical purity: 100%
- Lot/batch No.: OE10428
- Expiration date of the lot/batch: 2013-04-28
- Storage condition of test material: at room temperature (20 ± 5 °C)

Test animals

Species:

human

Strain:

other: EpidermTM, reconstructed three-dimensional human epidermis model (EPI-200)

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MatTek, In Vitro Life Science Laboratories, Bratislava, Slovakia

TEST METHOD
The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of human epidermis. It consists of organised basal, spinous and granular layers, and a multi-layered stratum corneum containing intracellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues are cultures on specially prepared cell culture inserts. Irritant materials are identified by their ability to penetrate the stratum corneum and to damage the underlaying cell layers which is determined through a decrease in cell viability as determined by MTT reduction assay.

ADAPTATION TO CELL CULTURE CONDITIONS
Tissues were inspected for viability and each three tissue inserts were transferred into three wells of each eight 6-well plates containing 900 µL assay medium per well. The plates were pre-incubated in an incubator for 1 h (37 °C, 5% CO2) before use. After pre-incubation, the other 3 wells of each plate were filled with 0.9 mL fresh assay medium and the remaining tissues were inserted into the respective plates. Then, all 6-well plates were incubated at 37 °C and 5% CO2 for a period of 18 h.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37
- CO2 gas concentration (%): 5.0

Test system

Type of coverage:

other: open (in vitro system)

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with Dulbecco’s phosphate buffered saline (DPBS) served as negative controls, positive controls were exposed to 5% SDS

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: SDS, 5% (v/v) in deionised water

Duration of treatment / exposure:

1 h

Observation period:

Not applicable. Post-treatment incubation period: 42 h

Number of animals:

Not applicable. The test was performed in triplicates for each treatment and control group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: the tissues were rinsed with DPBS to remove residual test substance.
- Time after start of exposure: 60 min
- Post-treatment incubation period: 42 h

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed 42 h after the incubation period. Therefore, tissues were incubated in 0.3 mL MTT solution (1 mg/mL in medium) for 3 h at 37 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed with DPBS for several times and subsequently thoroughly dried. For the extraction of the formazan product, each insert was placed into an empty pre-warmed 24-well plate and 2 mL of isopropanol was pipetted into each insert. After shaking the plate for 2 h at room temperature, the optical density of the solutions was measured at 570 nm wave length in a 96-well plate using a plate spectrophotometer.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls (DPBS). Time point: 1 h. Reversibility: other: reversibility: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

14.7

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls (5% SDS). Time point: 1 h. Reversibility: other: reversibility: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: cell viability (% of negative control)

Value:

99.4

Remarks on result:

other:

Remarks:

Basis: other: mean value of the test item (100%). Time point: 1 h. Reversibility: other: reversibility: not applicable. (migrated information)

In vivo

Other effects:

No further effects were observed.

Any other information on results incl. tables

Table 3. Results of MTT assay after 1 h exposure

	Negative control			Test item			Positive control
Tissue sample	1	2	3	1	2	3	1	2	3
OD570	1.466 1.277	1.113 1.087	1.096 1.088	1.298 1.179	1.164 1.161	1.140 1.146	0.212 0.209	0.210 0.212	0.211 0.210
OD570(mean)	1.329	1.057	1.049	1.196	1.120	1.100	0.168	0.168	0.168
OD570(mean values of replicates±%RSD)	1.145 ± 13.9			1.139 ± 4.4			0.168 ± 0.0
Viability (%)	100			99.4			14.7

OD = optical density; RSD = relative standard deviation

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified