Fatty acids, C16-18 (even numbered), aluminum salts

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 June - 6 June 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 13. April 2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

adopted 30. May, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), aluminum salts

EC Number:

939-582-4

Cas Number:

1471315-26-0

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

Fatty acids, C16-18 (even numbered), aluminum salts

Details on test material:

- Name of test material (as cited in study report): Fatty acids, tallow, hydrogenated, aluminium salts
- Physical state: white solid powder
- Analytical purity: 84.33%
- Impurities (identity and concentrations): ash content 9.17%; humidity 0.75%; free fatty acids 14.13%; water-soluble salts 0.61%; aluminium 4.13%
- Purity test date: 2012-02-14
- Lot/batch No.: C200752-001
- Expiration date of the lot/batch: 2014-02-14
- Storage conditions: at room temperature
- Other: CAS 91770-05-7

Test animals

Species:

human

Strain:

other: reconstructed human epidermis (EpiDerm)

Details on test animals or test system and environmental conditions:

TEST SKIN MODEL
- Source: MaTek (Epi-200)
- Adaptions to cell culture conditions: Upon receipt, the tissues were transferred into 6-well plates containing 900 µL prewarmed assay medium per well. The 6-well plates were incubated in a humidified incubator at 37 ± 1 °C, 5% CO2 for at least 1 h.

ENVIRONMENTAL 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 negative control tissue treated with aqua dest.

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 25 mg were applied directly atop the tissue, to ensure good contact the test item was moistened with 25 µL H2O

Duration of treatment / exposure:

3 and 60 min exposure

Observation period:

Not applicable

Number of animals:

Not applicable. Tests were performed in duplicate for each dose group.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing: after the incubation period, the inserts were washed carefully with PBS about 20 times.
- Time after start of exposure: 3 or 60 min, respectively

CELL VIABILITY MEASUREMENTS
- Method: MTT assay
- Details on method used: for viability testing, the inserts were placed in a prepared 24-well plate containing 300 µL MTT medium and further incubated for 3 h (37 °C, 5% CO2). After the incubation period, the MTT solutions were replaced by PBS for 3 times (washing) and afterwards the tissues were dried and transferred into extraction plates and covered with isopropanol. Extraction was carried out for 2 hours at room temperature on a shaker. Plates were placed for 15 ± 2 min on a plate shaker. The inserts were pierced with an injection needle after the incubation period to allow the extracts to run through the tissues into the corresponding wells. The inserts were discarded and the extraction plates again shaked for 15 minutes. Measurement of cell viability was carried out in 96-well plates. Per tissue, triplicates of 200 µL aliqouts were pipetted into 96-wells and absorption was measured at 550 nm without reference wavelength in a plate spectrophotometer (TECAN Infinite 200, TECAN Austria).

PRE-EXPERIMENTS
- To check the non-specific MTT-reducing capability of the test item 30 mg of the test item were mixed with 1 mL MTT medium and incubated for 1 h at room temperature. If the mixture turns blue/purple, the test item is presumed to have reduced MTT. Furthermore, the colouring potential of the test item in water was tested (25 mg test item mixed with 300 µL water).

EVALUATION CRITERIA
Corrosivity potential of the test item was predicted form the relative mean tissue viabilities compard to the negative control tissues concurrently treated with aqua dest. The substance is classified corrosive to skin if the tissue relative viability after 3 min of exposure is decreased below 50%. In addition, those test items classified as non-corrosive after 3 min (viability > 50%) are classified as corrosive if the relative mean tissue viability after 60 min treatment is decreased below 15% compared to the concurrent negative control tissue.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: negative control (aqua dest.)

Time point:

other: 15 min

Score:

100

Results 2

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: test material

Time point:

other: 3 min

Score:

90

Results 3

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: test material

Time point:

other: 60 min

Score:

114

Results 4

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: positive control (8 N KOH)

Time point:

other: 3 min

Score:

17

Results 5

Irritation parameter:

other: cell viability (% of negative control)

Basis:

other: positive control (8 N KOH)

Time point:

other: 60 min

Score:

9

Irritant / corrosive response data:

The cell viability after 3 min and 60 min exposure was greater than 50% and 15%, respectively.

Other effects:

No further effects were reported.

Any other information on results incl. tables

Table 1. Summary of results.

	Total OD550of 2 replicate tissues	Mean relative tissue viabilitiy[%]	SD tissue viability [%]
3 min
Negative Control PBS	2.112*	100	8.3***
Test Item	1.896	90	14.9***
Positive Control 5% SDS	0.355	17**	15.1***
60 min
Negative Control PBS	2.174*	100	0.8***
Test Item	2.479	114	27.7***
Positive Control 5% SDS	0.204	9	18.7***

*: mean OD550 > 0.8 of the negative control. Therefore, the validity criteria are fulfilled.

**: mean relative tissue viability of the 3 min positive control ≤ 30%. Therefore the validity criteria are fulfilled.

***: inter tissue viability difference < 30% for all dose groups. Therefore, the validity criteria are fulfilled.

OD: Absorption at 550 nm

SD: Standard deviation

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

According to Regulation (EC) No. 1272/2008, 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.