Phosphoric acid, mono- and bis(branched and linear pentyl) esters

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

The study was performed between 06 March 2012 and 08 March 2012.

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted on an analog substance in compliance with current OECD test guideline and in compliance with the GLP (for justification of read-across between phosphoric acid, mixture of pentyl ester and Reaction Mass of Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate, please refer to corresponding assessment report in Section 13).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other 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:

other: OECD guideline for the testing of chemicals 431

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Remarks:

2011-08-31

Test material

Test material information

Constituent 1

Reference substance name:

Reaction Mass of Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate

IUPAC Name:

Reaction Mass of Diisobutyl hydrogen phosphate and Isobutyl dihydrogen phosphate

Test material form:

other: liquid at room temperature

Details on test material:

- Name of test material (as cited in study report): Isobutyl acid phosphate (IBAP)

Test animals

Species:

other: reconstituted human epidermis model

Strain:

other: reconstituted human epidermis model

Details on test animals or test system 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.

- Type of wrap if used:
None used

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

Results

Results 1

Irritation / corrosion parameter:

other: other: Relative Mean Viability of cells

Remarks on result:

other:

Remarks:

Time point: 3 min exposure. Reversibility: other: Not applicable. Remarks: 103.7%. (migrated information)

Results 2

Irritation / corrosion parameter:

other: other: relative Mean Viability of cells

Remarks on result:

other:

Remarks:

Time point: 60 min exposure. Reversibility: other: Not applicable. Remarks: 11.1%. (migrated information)

Results 3

Irritation / corrosion parameter:

other: other: relative Mean Viability of cells

Remarks on result:

other:

Remarks:

Time point: 240 min exposure. Reversibility: other: Not applicable. Remarks: 4.1%. (migrated information)

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 OD₅₄₀ for the negative control treated tissues was 0.217. The negative control acceptance criterion was therefore satisfied.

Table1 : Mean OD₅₄₀ Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period	Mean OD540 of 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%

Applicant's summary and conclusion

Interpretation of results:

Category 1B (corrosive)

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.

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 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.