(1,2-dimethylpropyl)-5-methyl-pyrazole-4-carboxylic acid

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt Rheinland-Pfalz

Test material

Test material information

Constituent 1

Reference substance name:

(1,2-dimethylpropyl)-5-methyl-pyrazole-4-carboxylic acid

EC Number:

846-828-3

Cas Number:

1872341-39-3

Molecular formula:

C10H16N2O2

IUPAC Name:

(1,2-dimethylpropyl)-5-methyl-pyrazole-4-carboxylic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: L2018-137
- Expiration date of the Batch: 01 Feb 2021
- Purity: 99.6 % (tolerance ± 1.0 %)
- pH value: ca. 4 (undiluted test substance moistened with deionized water, determined in the lab prior to start of the GLP study)
- Physical state / color: Solid / yellowish

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

FORM AS APPLIED IN THE TEST (if different from that of starting material) : solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPI-200
- Tissue batch number: 28684

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: one washing step with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- Incubation time: 3 hours
- Spectrophotometer: Sunrise Absorbance Reader
- Wavelength: 570 nm
- Filter: measurement without reference filter

NUMBER OF REPLICATE TISSUES: two tissues per exposure time and test group (12 tissues per test)

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- killed tissues
- Procedure used to prepare the killed tissues (if applicable): freeze-killed at - 20 °C
- N. of replicates: two killed control tissues per exposure time were treated with the test substance and the negative control, respectively
- Method of calculation used: In case of direct MTT reduction by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the tissues treated with the test substance (mean corrected OD570 KC). Since killed tissues might still have a residual enzyme activity that is able to produce some formazan net, OD570 KC is calculated by subtracting the OD570 KC of the NC from the OD570 KC of the test substance. In case the net OD570 KC is greater than zero, it is subtracted from the respective mean OD570 to result in the mean corrected OD570 KC. The mean corrected OD570 KC represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: one

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50 %, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15 %.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is greater than or equal to 15 %

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8 N

Duration of treatment / exposure:

3 min and 1 h

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure period

Value:

95.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 h exposure period

Value:

105.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Table 2: Summary of results after 3 minutes exposure period

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	2.016	1.996	2.006
		Viability [% of NC]	100.5	99.5	100.0	0.7	0.7
	KC tissues	Mean OD570	0.114	0.092	0.103
		Viability [% of NC]	5.7	4.6	5.1	0.8	15.4
Test substance	Viable tissues	Mean OD570	1.995	1.849	1.922
		Viability [% of NC]	99.5	92.2	95.8	5.1	5.4
	KC tissues *	Mean OD570 KC NC corrected	0.011	0.000	0.006
		Viability [% of NC]	0.56	0.00	0.28
	Final relative mean viability of tissues after KC correction [% of NC]				95.5
PC	Viable tissues	Mean OD570	0.254	0.216	0.235
		Viability [% of NC]	12.7	10.8	11.7	1.3	11.4

*negative values are set to zero for further calculation

Table 3: Summary of results after 1 hour exposure period

Test substance identification			Tissue 1	Tissue 2	Mean	SD	CV [%]
NC	Viable tissues	Mean OD570	1.854	1.875	1.864
		Viability [% of NC]	99.4	100.6	100.0	0.8	0.8
	KC tissues	Mean OD570	0.085	0.092	0.088
		Viability [% of NC]	4.5	4.9	4.7	0.3	5.6
Test substance	Viable tissues	Mean OD570	1.941	2.030	1.986
		Viability [% of NC]	104.1	108.9	106.5	3.4	3.2
	KC tissues	Mean OD570 KC NC corrected	0.023	0.019	0.021
		Viability [% of NC]	1.2	1.0	1.1	0.1	11.9
	Final relative mean viability of tissues after KC correction [% of NC]				105.4
PC	Viable tissues	Mean OD570	0.097	0.106	0.101
		Viability [% of NC]	5.2	5.7	5.4	0.3	6.3

Table 4: Historic control data of NC and PC of skin corrosion test

		Exposure Time	Period	Mean	SD	Mean + 2 SD	Mean – 2 SD
NC	OD570	3 minutes	Jan 2017 – Jan 2019	1.728	0.216	2.160	1.295
		60 minutes		1.724	0.252	2.227	1.220
PC		3 minutes		0.222	0.071	0.363	0.081
		60 minutes		0.097	0.031	0.159	0.036
Relative viability [%]		3 minutes		13.0	4.2	21.3	4.6
		60 minutes		5.7	1.8	9.3	2.2

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

no indication of skin corrosion

Conclusions:

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.

Executive summary:

The potential of the test substance to cause dermal corrosion was assessed by a single topical application of ca. 25 μL bulk volume (about 7 mg) undiluted test substance to a reconstructed three-dimensional human epidermis model (EpiDerm™).

For the skin corrosion test, two EpiDerm™ tissues were incubated with the test substance for 3 minutes and 1 hour, each.

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation by using a colorimetric test. The reduction of mitochondrial dehydrogenase activity measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the epidermal tissues treated with the test substance is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The following results were obtained in the EpiDerm™ skin corrosion test:

The test substance is able to reduce MTT directly. Therefore, an additional MTT reduction control KC (freeze-killed control tissues) was introduced.

The final relative mean viability of the tissues treated with the test substance determined after an exposure period of 3 minutes was 95.5%, and it was 105.4% after an exposure period of 1 hour.

Based on the results observed and by applying the evaluation criteria, it was concluded that the test substance does not show a skin corrosion potential in the EpiDerm™ in vitro skin corrosion test under the test conditions chosen.