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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2017 - 16 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
20 July 2012
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium dihydrogen citrate
EC Number:
224-694-1
EC Name:
Ammonium dihydrogen citrate
Cas Number:
4450-94-6
Molecular formula:
C6H8O7.H3N
IUPAC Name:
azanium; 3-carboxy-3,5-dihydroxy-5-oxopentanoate
Constituent 2
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
oxidane
Test material form:
liquid
Details on test material:
Identification: Ammonium di hydrogen citrate
CAS number: 4450-94-6
Appearance: Clear colourless liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM, 0.38 cm^2
- Tissue batch number: 17-EKIN-041
- Twenty five μL of the undiluted test substance was added into 12-well plates on top of the skin tissues.
- The test item was applied topically to the corresponding tissues ensuring uniform covering.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.5 - 37.2°C

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 25 μL
Duration of treatment / exposure:
15-Minute exposure period
Duration of post-treatment incubation (if applicable):
42 hours post-exposure incubation period.
Number of replicates:
A total of 9 tissues were used: Triplicate tissues were treated with: test substance, positive control or negative control.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
110
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: The relative mean tissue viability compared to the negative control tissues (100%).
Other effects / acceptance of results:
OTHER EFFECTS:
- Colour interference with MTT and Direct-MTT reduction: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 10%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was < 7%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
According to Regulation (EC) No. 1272/2008.
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that the test substance is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model) performed according to OECD439 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to 0.38 cm^2 cultured skin (25 μL). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 10% whereas the test substance showed cell viability of 110%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that the test substance is not irritating in the in vitro skin irritation test and the substance does not have to be classified for skin irritation according to Regulation (EC) No. 1272/2008.