Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 Dec 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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)

Version / remarks:

adopted in 2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany

Test material

Test material information

Constituent 1

Reference substance name:

2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid

EC Number:

940-877-5

Cas Number:

2241455-89-8

Molecular formula:

C9H12N2O4

IUPAC Name:

2-(3,4-dimethyl-1H-pyrazol-1-yl)butanedioic acid; 2-(4,5-dimethyl-1H-pyrazol-1-yl)butanedioic acid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDerm, reconstructed three-dimensional human epidermis (EPI-200)

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

TEST SKIN MODEL
- Source: MatTek Corporation, Ashland MA, USA

TEST METHOD
EpiDermTMSCIT (EPI-200):
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. Irritant 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 6-well plates containing 900 µL prewarmed assay medium per well and preincubated in a humidified incubator for at least 1 h (37 ± 1 °C, 5% CO2) before use.

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

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

CELL VIABILITY MEASUREMENTS
For determining alterations in cell viability, MTT reduction assays were performed. Therefore, tissues were incubated in 300 µL prewarmed MTT solution for 3 h at 37 ± 1 °C and 5% CO2. After aspiration of the MTT solution, tissues were washed 3 times in phosphate buffered saline followed by tissue drying. Extraction of the formazan product was carried out in 2 mL isopropanol. The optical density was measured at 550 nm wave length in a plate spectrophotometer.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 25 mg
- Concentration: 100% (moistened with 25 µL water)

POSITIVE CONTROL SUBSTANCE:
- Positive control substance: Potassium hydroxide (8 N, CAS 1310-58-3, Lot 10357004)

Duration of treatment / exposure:

3 and 60 min

Number of replicates:

The test was performed in duplicates for each test or control group and treatment period (3 and 60 min).

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min of exposure

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min of exposure

Value:

88

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

After treatment with the test item the viability was 93% and 88% after treatment for 3 and 60 min, respectively. Therefore the test item is considered to be not corrosive.

Any other information on results incl. tables

Table 1: MTT assay after 3 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD550	2.088 2.085 2.082	1.947 1.985 1.978	0.309 0.315 0.312	0.269 0.271 0.269	2.001 1.907 1.878	1.803 1.859 1.818
OD550(mean)	2.085	1.970	0.312	0.270	1.929	1.827
SD	0.003	0.020	0.003	0.001	0.064	0.029
OD550(mean values of replicates)	2.028		0.291		1.878
Viability (%)	100		14		93
Mean inter tissue viability difference (%)	5.7		14.5		5.4

 

Table 2: MTT assays after 60 min exposure

	Negative control		Positive control		Test item
Tissue sample	1	2	1	2	1	2
OD550	2.032 1.954 1.976	2.014 1.992 1.974	0.114 0.117 0.115	0.119 0.129 0.121	1.857 1.754 1.762	1.742 1.717 1.736
OD550(mean)	1.987	1.993	0.115	0.123	1.791	1.732
SD	0.040	0.020	0.002	0.005	0.057	0.013
OD550(mean values of replicates)	1.990		0.119		1.761
Viability (%)	100		6		88
Mean inter tissue viability difference (%)	0.3		6.4		3.4

 

Table 3: Historical data

	mean	SD	n
OD550of Negative Controls (3 min)	1.731	0.176	25
OD550of Negative Controls (60 min)	1.748	0.192	25
Relative tissue viability (%) of positive controls (3 min)	19.0	5.958	24
Max. inter tissue viability difference (%)	13.9	6.627	144

 

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive (Skin Irrit. 2 or not classified according to Regulation (EC) No 1272/2008)

Conclusions:

There is regulatory acceptance in the EU that a substance can be considered corrosive 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.