Dilithium azelate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

21 April 2015 to 24 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant, guideline study, available as an unpublished report.

Justification for type of information:

For read across justification, see Section 13 of IUCLID

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium adipate

EC Number:

242-449-7

EC Name:

Dilithium adipate

Cas Number:

18621-94-8

Molecular formula:

C6H8O4.2Li

IUPAC Name:

dilithium adipate

impurity 1

Reference substance name:

Adipic acid

EC Number:

204-673-3

EC Name:

Adipic acid

Cas Number:

124-04-9

Molecular formula:

C6H10O4

IUPAC Name:

adipic acid

Test material form:

solid

Details on test material:

- Batch number: A049-99
- Expiry date: 06 March 2018
- Storage condition of test material: At room temperature protected from light

Test animals

Species:

other: EPISKIN-SM Human epidermis model (Lot no: 15-EKIN-016)

Strain:

other: Not applicable

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:

unchanged (no vehicle)

Controls:

other: Not applicable.

Amount / concentration applied:

- Treatment group: Test carried out in triplicate. 12.4 to 16.7 mg of the test item was applied topically with a small glass weight boat, ensuring an even covering, to the epidermis surface which had previously been moistened with 5 µL sterile, distilled water to improve contact between the solid test item and the epidermis.
- Negative control: Triplicate tissues treated with 25 µL Phosphate buffered saline (PBS)
- Positive control: Triplicate tissues. 25 µL Sodium Dodecyl Sulphate (SDS) at 5% w/v aqueous solution spread over entire surface of the epidermis using a pipette tip with the process being repeated after 7 minutes.

Duration of treatment / exposure:

- Treatment period: 15 minutes
- Post-exposure incubation: At the end of the exposure period, tissues were rinsed with PBS to remove any residual test item. The rinsed tissues were dried carefully and transferred to a new well on 2 mL pre-warmed maintenance medium, and incubated for 42 hours, at 37°C and 5 % CO2 in air.

Observation period:

Not applicable.

Number of animals:

Not applicable.

Details on study design:

A test substance may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test substance is present on the tissues when the MTT viability test is performed.
Dilithium adipate was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 508277). Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that Dilithium adipate did not interfere with the MTT endpoint.

Preincubation:
The tissues were transferred to 12-well plates and preincubated with pre-warmed Maintaince medium for 26 hours at 37°C, 5 % ± 0.5 Co2 in air.

Application/Treatment of the test substance
The test was performed on a total of 3 tissues per test substance together with negative and positive controls. The skin was moistened with 5 μL Milli-Q water and the solid test substance (12.4 to 16.7 mg; with a small glass weight boat) was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5% SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time. After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with PBS to remove residual test substance. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37°C.

Cell vaibility measurement
Cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-medium (0.3 mg/mL). The tissues were incubated for 3 h at 37°C. After incubation the tissues were placed on blotting paper to dry. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in prelabeled microtubes and extracted with 500 μL isopropanol. Tubes were stored at room temperature and protected from light for 4 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test substance was classified according to remaining cell viability following exposure of the test substance.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes

Value:

103

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Dilithium adipate compared to the negative control tissues was 103%. The mean optical density measured at 570 nm was 1.026 with standard deviation of 0.099.
The test item is considered to be non-irritant using the EPISKIN-SM (TM) human epidermis model.
- Positive control: The relative mean tissue viability was 8 % relative to the negative control. The mean optical density was 0.083 and the standard deviation was 0.029.
- Negative control: The mean optical density was 0.992 and the standard deviation was 0.108.

Any other information on results incl. tables

Table 1. Mean absorption (OD570) values and percentage viabilities for the negative control, positive control and test items.

Item	OD570of tisues	Mean OD570 of triplicate tissues	± SD of OD570	Relative mean viability (%)
Negative control item	0.917	0.992	0.108	100
	0.944
	1.116
Positive control item	0.076	0.083	0.029	8
	0.115
	0.058
Dilithium adipate	1.079	1.026	0.099	103
	0.912
	1.087

OD - optical density

SD - standard deviation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It is concluded that this test is valid and that Dilithium adipate is non-irritant in the in vitro skin irritation test under the experimental conditions described in the report.

Executive summary:

The purpose of the test was to evaluate the skin irritation potential of the Dilithium adipate using the EPISKIN-SM reconstructed human epidermis model according to the OECD 439 guideline. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Dilithium adipate compared to the negative control tissues was 103%. Since the mean relative tissue viability for Dilithium adipate was above 50% after 15 ± 0.5 minutes treatment Dilithium adipate is considered to be non-irritant.