4-pyridylamine

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-pyridylamine

EC Number:

207-987-9

EC Name:

4-pyridylamine

Cas Number:

504-24-5

Molecular formula:

C5H6N2

IUPAC Name:

nickel

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: human epidermal keratinocytes (NHEK)

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

other: PBS were applied to the epidermal surface in order to improve the contact between the powder and the epidermis.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.

Duration of post-treatment incubation (if applicable):

The plates were post-incubated at 37 +- 1°C, 5.0% CO2, humidified to 95%, for 24 +/- 2 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2.6

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Name	Negative Control			Positive Control			Test Item
Tissue	1	2	3	1	2	3	1	2	3
Absolute OD570	1.956	1.874	1.856	0.096	0.102	0.099	0.091	0.089	0.092
	1.969	1.899	1.889	0.097	0.103	0.102	0.095	0.091	0.094
OD570(Blank Corrected)	1.913	1.832	1.814	0.053	0.059	0.057	0.049	0.046	0.049
	1.926	1.856	1.846	0.055	0.061	0.059	0.052	0.049	0.051
Mean OD570of the Duplicates (Blank Corrected)	1.920	1.844	1.830	0.054	0.060	0.058	0.050	0.048	0.050
Total Mean OD570of 3 Replicate Tissues (Blank Corrected)	1.865*			0.057*			0.049*
SD OD570	0.048			0.003			0.002
Relative Tissue Viability [%]	103.0	98.9	98.1	2.9	3.2	3.1	2.7	2.5	2.7
Mean Relative Tissue Viability [%]	100.0			3.1**			2.6
SD Tissue Viability [%]***	2.6			0.2			0.1
CV [% Viabilities]	2.6			5.4			3.1

The potential of the test item to induce skin irritation was analysed by using the three-dimensional human epidermis model EpiDerma(MatTek) comprising a reconstructed epidermis with a functional stratum corneum.

In the present study 4-Aminopyridine was applied topically to the EpiDerma tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned red/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.

The mixture of 25 mg of the test item per 300 µl aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (2.6%) after 60 min treatment and 42 h post-incubation.

The controls confirmed the validity of the study. The mean absolute OD₅₇₀ of the three negative control tissues was³ 0.8 and ≤ 2.8. The mean relative tissue viability (% negative control) of the positive control was£ 20% (3.1%). Standard deviation of viability of replicate tissues of all dose groups was≤ 18% (0.1% – 2.6 %).

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

„In this study under the given conditions the test item showed irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was ≤ 50%. However, it cannot be stated whether the substance is either corrosive (Cat. 1) or irritant (Cat. 2) to the skin. Therefore, additional information on skin corrosion potential is needed.

Executive summary:

In the present study the skin irritant potential of 4-Aminopyridine was analysed.The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404,[7]) to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation periodand compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned red/purple. The coloured test material or MTT reducing substance was classified as “Irritant” i.e. mean tissue viability is < 50%. Therefore, no correction procedures were necessary.

The mixture of 25 mg of the test item per 300 µl aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 50% (2.6%) after 60 min treatment and 42 h post-incubation.