4-pyridylamine

Eye irritation

Administrative data

Endpoint:

eye 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 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Deviations: Concerning: Provided Materials, study plan, p. 11
Study Plan:1x vial Methyl acetate, CAS No. 79-20-9 (positive control)
Reason:Methyl acetate was not provided with the kit.

Deviations:

yes

Remarks:

This deviation did not influence the quality or integrity of the present study.

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

Test animals / tissue source

Species:

human

Details on test animals or tissues and environmental conditions:

This test uses the three-dimensional RhCE EpiOcular™ (MatTek). It consists of normal, human-derived epidermal keratinocytes and mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium. The MatTek EpiOcular™ model has been widely used as a research and testing model for many years.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 50 mg (83.3 mg/cm2) of the test item were applied directly atop the EpiOcular™ tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue.

Duration of treatment / exposure:

for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air

Duration of post- treatment incubation (in vitro):

18 ± 0.25

Number of animals or in vitro replicates:

2

Results and discussion

In vitro

Results

Irritation parameter:

other: mean tissue viability [%]

Run / experiment:

mean

Value:

1.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Value Cut off pass/fail
Mean Absolute OD570 nm NK 1.735 0.8 < NK < 2.5 pass
Mean Relative Viability PC [%] 28.4 < 50% pass
Max. Difference of % Viability [%] 1.5 < 20% pass

Any other information on results incl. tables

The potential of the test item to induce eye irritation was analysed by using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. In the present study 4-Aminopyridine was applied topically to the EpiOcularätissue for 6 h followed by 25 min post-soaking incubation after removal of the test item. After a 18 h post-treatment period cytotoxic effects were determined via MTT reduction assay. Ocular irritation potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with aqua dest. The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The test material turned blue/purple and the mixture turned purple/red. Sincethe mean relative tissue viability of the test item treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test item has to be classified as irritant in any case. The mixture of 50 mg test item per 1 mL A. dest. and per 2 mL isopropanol showed no colouring as compared to the solvent. Therefore, NSC_living equalled 0%. The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (1.7%). The controls confirmed the validity of the study. The mean absolute OD₅₇₀of the two negative control tissues was> 0.8 and < 2.5 (1.735). The mean relative tissue viability (% negative control) of the positive control was < 50% (28.4%). The maximum inter tissue difference of replicate tissues of all dose groups was < 20% (1.5%).

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed irritant effects. The test item is classified as “irritant“ in accordance with UN GHS “Category 1” or “Category 2”.

Executive summary:

In the present study the eye irritating potential of 4-Aminopyridine was analysed. Since irritant substances are cytotoxic to the corneal epithelium after a short time exposure the cytotoxic effects of the test item on EpiOcular, a reconstituted three-dimensional human corneal epithelium model, were determined. 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 6 h exposure period and 18 h post-treatment period and compared to those of the concurrent negative controls.

The mixture of 50 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The test material turned blue/purple and the mixture turned purple/red. Since the mean relative tissue viability of the test item treated tissues (TM) was below the 60% threshold value no killed tissue controls were performed, since the test item has to be classified as irritant in any case.

The mixture of 50 mg test item per 1 mL A. dest. and per 2 mL isopropanol showed no colouring as compared to the solvent.Therefore, NSC_livingequalled 0%.

The test item showed irritant effects. The mean relative tissue viability (% negative control) was ≤ 60% (1.7%).