Alcohols, C16-18, ethoxylated, phosphates

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug - 31 Aug 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The department of health of the government of the United Kingdom.

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C16-18, ethoxylated, phosphates

EC Number:

500-295-0

EC Name:

Alcohols, C16-18, ethoxylated, phosphates

Cas Number:

106233-09-4

Molecular formula:

UVCB

IUPAC Name:

Alcohols, C16-18 (even numbered) ethoxylated, phosphates, mono- and diesters

Details on test material:

- Name of test material (as cited in study report): Trade name (CAS 106233-09-4)
- Substance type: UVCB
- Physical state: off white solid block
- Analytical purity: >95%
- Lot/batch No.: 533935
- Expiration date of the lot/batch: 2013-09-05
- Storage condition of test material: room temperature in the dark

Test animals

Species:

human

Strain:

other: EPISKIN, three-dimensional reconsructed human epidermis model

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: SkinEthic Laboratories, Lyon, France

TEST METHOD
The model represents a reconstructed three-dimensional skin model based on normal human-derived epidermal keratinocytes which have been cultured to form a multilayered epidermis including basal, spinous and granular layers, and a multi-layered stratum corneum. Corrosive 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
Upon receipt, tissues were transferred into 12-well plates containing 2.2 mL prewarmed maintenance medium per well and preincubated in a humidified incubator for 24 h (37 ± 1 °C, 5% CO2) before use. After 24 h the medium was refreshed and the tissues were incubated for further 24 h.

INCUBATION CONDITIONS (INCUBATOR)
- Temperature (°C): 37 ± 1
- CO2 gas concentration (%): 5
- Humidity: maximum

Test system

Type of coverage:

open

Preparation of test site:

other: intact reconstructed human epidermis

Vehicle:

unchanged (no vehicle)

Controls:

other: concurrent control tissues treated with 0.9% saline; positive controls were exposed to glacial acetic acid

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 20 mg

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: glacial acetic acid

Duration of treatment / exposure:

3, 60, and 240 minutes

Observation period:

not applicable

Number of animals:

Not applicable. The test was performed in duplicates for each test or control group and treatment period (3, 60, and 240 min)

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: The test item was washed from the skin surface with phosphate buffered saline with Ca2+ and Mg 2+.
- Time after start of exposure: 3, 60, and 240 min

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 2.2 mL prewarmed MTT solution (0.3 mg/mL MTT) for 3 h at room temperature (under light protection). At the end of the 3-h incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was taken. The epidermis was separated from the collagen matrix and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL tubes containing 850 µL isopropanol. The optical density (OD) was measured at 540 nm wave length in a plate spectrophotometer.

Test for direct MTT reduction:
To identify if the test item may interfere with MTT a pre-test was performed, using a solution of MTT (2.2 mL; 0.3 mg/mL MTT) with 20 mg test item. The solution was incubated at room temperature for 3 h. Untreated MTT solution was used as a control.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

other: other: cell viability (%)

Value:

100

Remarks on result:

other:

Remarks:

Basis: other: mean value of negative controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Results 2

Irritation / corrosion parameter:

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

Value:

12.8

Remarks on result:

other:

Remarks:

Basis: other: mean value of positive controls. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Results 3

Irritation / corrosion parameter:

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

Value:

67.9

Remarks on result:

other:

Remarks:

Basis: other: mean value of test item. Time point: 240 min. Reversibility: other: not applicable. (migrated information)

Results 4

Irritation / corrosion parameter:

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

Value:

79.5

Remarks on result:

other:

Remarks:

Basis: other: mean value of test item. Time point: 60 min. Reversibility: other: not applicable. (migrated information)

Results 5

Irritation / corrosion parameter:

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

Value:

92.3

Remarks on result:

other:

Remarks:

Basis: other: mean value of test item. Time point: 3 min. Reversibility: other: not applicable. (migrated information)

In vivo

Irritant / corrosive response data:

No direct interference of MTT and the test item was observed. The MTT solution containing the test item did not turn blue.
The relative mean viability of the test item treated tissues was 67.9, 79.5, and 92.3% for the 240, 60, and 3 min exposure period, respectively. Therefore the test item was considered to be non-corrosive to the skin. The acceptance criteria of the negative and positive control were satisfied. A tissue viability of 12.8% was observed after 240 min exposure to the positive control substance. The mean OD540 for the negative control treated tissues was 0.156. Thus, the acceptance criterion was satisfied.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive (Skin Corrosive Cat.1A) based on a positive result in the human epidermis model test. Negative in vitro corrosivity responses are not conclusive with respect to non-classification or classification as irritant and shall therefore be subject to further evaluation.