dipropyl cyclohexane-1,2-dicarboxylate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th July 2015 -28th August 2015

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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:

Reconstructed Human Epidermis Test Method, Guideline 439, 26 July 2013.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: In Vitro Skin Irritation Test: Human Epidermis Model EpiSkin™, EpiSkin™ Skin Irritation Test15 min - 42 hours – February 2009 Version 1.8, supplied by L’Oreal (leading laboratory in the validation of the test for ECVAM).

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

EC Number:

805-172-8

Cas Number:

65646-25-5

Molecular formula:

C14 H24 O4

IUPAC Name:

1,2-dipropyl cyclohexane-1,2-dicarboxylate

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): CHP
- Physical state: Colourless Liquid
- Analytical purity: 98.6%
- Lot/batch No.:ST111209
- Expiration date of the lot/batch:31 December 2016
- Storage condition of test material:Room temperature (ca. 20°C), in the dark, under gaseous nitrogen

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The EpiSkin™ Skin Irritation Test15 min - 42 hours using reconstructed human epidermis skin constructs supplied by SkinEthic Laboratories, Lyon, France, was accepted as a replacement to the in vivo Draize Skin Irritation Test, by the European Centre for the Validation of Alternative Methods (ECVAM) in a statement dated 27 April 2007.

The test involves the application of the test substance for 15 minutes to the EpiSkin™ threedimensional human skin model. The model consists of normal, human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type 1
matrix coated with type IV collagen. After 13 days in culture a multilayered, highly differentiated model of the human epidermis with a functional multi-layered stratum corneum has formed. The epidermis surface area supplied is 0.38cm2. The EpiSkin™ kits include assay medium, maintenance medium, 12 well plates and the tissues which are shipped on nutritive agar.

The principle of the assay is that irritant substances are sufficiently cytotoxic to cause cell damage in the cell layers. The cell viability is determined bymitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify
irritant and non-irritant substances. The test includes acceptance criteria for both negative and positive controls.

Control samples:

other: The negative control was sterile Dulbecco’s Phosphate Buffered Saline (DPBS) with magnesium and calcium. The positive control was 5% Sodium Dodecyl Sulphate (SDS) in purified water.

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit):The test substance, CHP, a colourless liquid, positive and negative controls were in liquid
form and were applied by dispensing a volume of 10 micro litres over each tissue using a positive displacement pipette.

Duration of treatment / exposure:

After incubation of at least 24 hours in maintenance medium, triplicate tissues were dosed for 15 +/- 0.5 minutes with the test substance, negative orpositive control at room temperature. A maximum of four samples were applied in a block with a minimum of 1 minute intervals between each application of substance. On application of 10 μL, the positive control was spread over the tissue for approximately 30 seconds and then respread with a curved flat spatula after 7 minutes application time.

After 15 +/- 0.5 minutes, each tissue was rinsed with 25 mL sterile Dulbeccos Phosphate
Buffered Saline (DPBS) to remove residual test substance. Inserts were then blotted on absorbent paper to remove remaining DPBS. Each insert was then transferred to a well containing 2 mL maintenance medium and incubated for 42 +/- 1 hour at 37 +/- 2°C in a humidified atmosphere of 5% CO2 in air.

After 42+/- 1 hour, each insert was transferred to a well containing 2 mL of 0.3 mg/mL MTT and incubated for 3 hours+/- 5 minutes at 37 +/- 2°C in a humidified atmosphere of 5% CO2 in air.

At the end of 3 hours +/- 5 minutes, the triplicate inserts were blotted on absorbent paper. The epidermis was removed from the insert using a biopsy punch, the epidermis separated from the collagen matrix using forceps and both parts placed in a micro-tube.

When all tissues had been punched, the tissues were vortexed with 500 μL of acidic isopropanol (0.04 N HCl final concentration).

The tissues were extracted over 4 hours, protected from light at room temperature (vortexing after approximately 2 hours).
After formazan extraction, duplicate 200 μL aliquots of the extractant from each tube were pipetted into the wells of flat-bottomed 96-well plates. Theextractant was mixed by vortexing prior to taking the aliquots. The absorbance was read at 540 nm, with six wells containing acidified isopropanol (0.04 N HCl final concentration) as a blank.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other: other: Mean tissue viability

Value:

107.6

Remarks on result:

other:

Remarks:

Basis: other: Episkin. Reversibility: no data. (migrated information)

Any other information on results incl. tables

Assessment of viability

The mean Optical Density (OD) for the six replicate blanks was subtracted from the individual substance and control tissues OD.

The viability of each tissue was expressed as a percentage of the mean negative control value.

Assay acceptance criteria

Negative control

The OD from the negative control tissue in the MTT assay is an indicator of tissue viability after the shipping and storage procedure and under the specific conditions of the assay. The mean absorbance of the triplicate negative control values should be >=0.6 and ≤1.5. The Standard Deviation (SD) value of the % viability should be ≤18.

Positive control

The OD of the positive control is an indicator of the sensitivity of the tissues. The mean viability should be ≤40% of the negative control and the SD ≤18%.

Data interpretation - Prediction model

If the mean tissue viability was equal to or less than 50% of the negative control value, the sample was classed as Category 2 (GHS classification).

Criteria for in vitro interpretation	Globally Harmonized System
Mean tissue viability is ≤ 50 %	Category 2
Mean tissue viability is > 50 %	No category

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.

Executive summary:

The objective of this test was to assess the skin irritation potential, in vitro, of the test substance, CHP.

The test substance was applied to EpiSkin™ human epidermis skin constructs. The constructs consisted of normal, human-derived epidermal keratinocytes, which had been cultured to form a multilayered, highly differentiated model of the human epidermis with a functional multilayered stratum corneum. The constructs were treated with the neat test substance for 15 minutes. After rinsing of the test substance the constructs were incubated for 42 hours. The cell viability was determined by mitochondrial dehydrogenase activity, assessed by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a soluble, coloured, formazan product. The prediction model uses the percentage viability values (compared to negative control viability) to identify irritant and non-irritant substances.

This assay was valid with negative and positive controls showing results within the acceptable range.

The test substance, CHP, elicited a mean tissue viability of 107.6 ± 3.6% and was predicted as non-irritant to the skin, classification, No category.