Diisobutyl phthalate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 June - 04 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study. Available as an unpublished report. Acceptable without restrictions.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 431 "In Vitro Skin Corrosion: Human Skin Model Test" (adopted 13 April 2004)

Deviations:

no

Principles of method if other than guideline:

Test method used the EPISKIN™ in vitro Reconstituted Human Epidermis (RHE) Model after treatment periods of 3, 60, and 240 minutes.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diisobutyl phthalate

EC Number:

201-553-2

EC Name:

Diisobutyl phthalate

Cas Number:

84-69-5

Molecular formula:

C16H22O4

IUPAC Name:

1,2-bis(2-methylpropyl) benzene-1,2-dicarboxylate

Details on test material:

- Name of test material (as cited in study report): Diisobutyl-phthalate
- Physical state: clear colourless liquid
- Storage condition of test material: room temperature in the dark

Test animals

Species:

human

Strain:

other: not applicable

Details on test animals or test system and environmental conditions:

Human skin tissue used in in vitro test - EPISKIN Model Kit 0.38 cm2.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37 °C

Test system

Type of coverage:

other: Universal coverage of tissue sample in vitro

Preparation of test site:

other: not applicable

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL
- Concentration (if solution): Liquid test material used as supplied.

Duration of treatment / exposure:

3, 60, and 240 minutes.

Observation period:

Assessment of effects required complicated procedure, see below.

Number of animals:

Duplicate epidermis samples for each exposure and duration.

Details on study design:

PRE-TEST
An assessment of the test material's potential to reduce MTT (a reaction which would have invalidated the main assessment procedure) was first conducted.
50 µL of test material was added to 0.3 mg/mL MTT solution and incubated in the dark for 3 hours. Untreated solution was used as a control. A colur change to blue/purple relative to the control was observed for as this was indicative of a reduction.

MAIN TEST

2.2 mL of assay medium was pipetted into 2 wells fo the second and third columns of a 12-well plate. Tissues were transferred to the second colum.
Duplicate tissues were treated with the test material (50 µL) for the alloted time periods with negative and postive controls being treated with 0.9% w/v NaCl solution and glacial acetic acid respectively.
At the end of the exposure period tissues were removed and rinsed to remove the test material.
Tissues were then transferred to MTT filled wells of the plate and incubated for 3 hours ± 5 minutes at room tempterature then placed onto absorbant paper to dry.Tissues were then examined for MTT staining, and then by total biospy - the epidermis removed from the collagen matrix and both parts placed into 1.5 mL micro tubes containing 850 µL of acidified isopropanol. Tubes were plugged, mixed thoroughly and stored overnight at room temperature to extract formazan crystals out of the MTT-loaded tissues.
After this, tubes were mixed thoroughly on a vortex mixer to produce a homogenous coloured solution. Duplicate 200 µL samples were taken and the optical denisty measured (quantitative viability analysis) at 540 nm using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: OD540

Value:

0.202

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 3 minutes. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: OD540

Value:

0.214

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60 minutes. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other:

Value:

0.183

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 240 minutes. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

The test material is concluded to be Non-Corrosive to the skin.

Any other information on results incl. tables

The pre-test showed no reduction of MTT by the test material, thus the results of the test are not invalid.

Table 1. Mean OD540 Values and Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material	Exposure Period (minutes)	Mean OD540 of duplicate tissues	Relative mean viability (%)
Negative Control Material	240	0.099	Mean viability of negitive control tissues is set to 100%
Positive Control Material	240	0.018	18.2
Test Material	240	0.183	184.8
	60	0.214	216.2
	3	0.202	204

Applicant's summary and conclusion

Interpretation of results:

other: Non-Corrosive to the skin

Remarks:

Criteria used for interpretation of results: not specified

Conclusions:

Diisobutyl phthalate is non-corrosive to the skin.

Executive summary:

In an in vitro assay, treatment of skin tissues with diisobutyl phthalte was not observed to be corrosive. The test was conducted according to standard guidelines and GLP and met the validity criterea.