Registration Dossier

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was performed between 06 March 2012 and 08 March 2012.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD guideline for the testing of chemicals 431
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2011-08-31

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Viscous liquid stored at room temperature
Details on test material:
- Name of test material (as cited in study report): Isobutyl acid phosphate (IBAP)

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: reconstruted Human Epidermis
Cell source:
other: Culture

Test animals

Species:
other: reconstituted human epidermis model
Strain:
other: reconstituted human epidermis model
Details on test animals and environmental conditions:
Not applicable

Test system

Type of coverage:
other: Topical
Preparation of test site:
other: Not applicable
Vehicle:
other: No vehicle used
Controls:
no
Amount / concentration applied:
TEST MATERIAL

- The test item was applied neat.

- Amount(s) applied (volume or weight with unit):
50 µl of the test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues.

- Concentration (if solution):
The test item was used as supplied.

VEHICLE
No vehicle used
Duration of treatment / exposure:
3, 60 or 240 minutes
Observation period:
Not applicable
Number of animals:
Not applicable
Details on study design:
TEST SITE
- Area of exposure:
50 µl of the test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues.

- % coverage:
The test item was applied topically to the corresponding tissues ensuring uniform covering.

REMOVAL OF TEST ITEM
- Washing (if done):
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item.

- Time after start of exposure: 3, 60 or 240 minutes

SCORING SYSTEM:
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute treatments, compared to the mean of the negative control tissues. The relative mean viabilities were calculated in the following way:

mean OD540 of test material / mean OD540 of negative control x 100 = Relative mean tissue viability (percentage of negative control)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes
Value:
103.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes
Value:
11.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 minutes
Value:
4.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

In vivo

Irritant / corrosive response data:
The relative mean viability of the test material treated tissues was as follows:
240 minutes exposure: 4.1%
60 minutes exposure: 11.1%
3 minutes exposure: 103.7%
Other effects:
No

Any other information on results incl. tables

Direct MTT Reduction:

The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

Quality Criteria:

The relative mean tissue viability for the positive control treated tissues was 8.3% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD540for the negative control treated tissues was 0.217. The negative control acceptance criterion was therefore satisfied.

Table 7.3.1/1:Mean OD540Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item

Exposure Period

Mean OD540of duplicate tissues

Relative mean viability (%)

Negative Control Item

240 Minutes

0.217

100*

Positive Control Item

240 Minutes

0.018

8.3

Test Item

240 Minutes

0.009

4.1

60 Minutes

0.024

11.1

3 Minutes

0.225

103.7

*= The mean viability of the negative control tissues is set at 100% Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate (IBAP)

Applicant's summary and conclusion

Interpretation of results:
Category 1B (corrosive) based on GHS criteria
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
As viability of cells is higher than 35% after 3 minutes exposure but lower than 35% after 1 hour exposure the test item is classified as corrosive to the skin, Skin Corr. 1B (H314) according to the Regulation (EC) 1272/2008 (CLP) and as C, R34 according to the Directive 67/548/EEC under the test conditions of this study.
Executive summary:

In an in vitro skin corrosion study performed according to the OECD guideline 431 and in compliance with the GLP, Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate" (purity 96%) was applied undiluted to Reconstructed Human Epidermis (RHE) model (Episkin TM model kit 0.38cm²).

Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. At the end of the exposure period each tissue was rinsed from the test item before each tissue was taken for MTT-loading. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.  At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre‑labelled 96‑well plate. The optical density (OD) was measured at 540 nm (OD540). 0.9 % (w/v) Sodium Chloride Solution was used as the negative control. Glacial Acetic Acid was used as the positive control.

The relative mean tissue viability for the positive control treated tissues was 8.3% relative to the negative control treated tissues following the 240-minute exposure period. The positive control acceptance criterion was therefore satisfied. The mean OD540for the negative control treated tissues was 0.217. The negative control acceptance criterion was therefore satisfied.

Percentage of cell viability (MTT reduction in the test item treated tissues relative to negative control tissues) was determined for each exposure period. The relative mean viability of the test item treated tissues was 103.7; 11.1 and 4.1% for exposure period of 3, 60 and 240 min, respectively.

As viability of cells is higher than 35% after 3 minutes exposure but lower than 35% after 1 hour exposure the Reaction Mass of "Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate" is classified as corrosive to the skin, Skin Corr. 1B (H314) according to the Regulation (EC) 1272/2008 (CLP) and as C, R34 according to the Directive 67/548/EEC.

This study is considered as acceptable as it satisfies the main criteria of OECD guideline No. 431.