4-pyridylamine

Endpoint summary

Description of key information

The in vitro study results on skin irritation/corrosion showed irritant and corrosive effects and therefore the substance is classified as “corrosive“ in accordance with a combination of optional UN GHS sub-categories 1B.

The in vitro study on eye irritation was positive and due to the corrosion effects in the skin corrosion study the substance is classified as "corrosive" in accordance with UN GHS Category 1

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Justification for test system used:

The EpiDerm Skin Model is a well-established organotypic, three-dimensional model of the human epidermis and is used for in vitro experiments since many years. It is known for its similarity to human skin.
This in vitro method allows the identification of corrosive and non-corrosive substances and mixtures in accordance with UN GHS. It further allows a partial sub-categorisation of corrosives in optional sub-category 1A or a combination of optional sub-categories 1B and 1C.

Vehicle:

unchanged (no vehicle)

Details on test system:

25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. To ensure good contact with the skin the test item was moistened with 25 µL H2O. The test item was spread to match size of the tissue.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item.

Duration of treatment / exposure:

60 min treatment and 3 min treatment.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 3 min Experiment

Value:

80.7

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean 60 min Experiment

Value:

9.4

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

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

In the present study 4-Aminopyridine was applied topically to the EpiDerma tissue for 3 min and 60 min followed by immediate determination of cytotoxic effects via MTT reduction assay.

Corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after both treatment periods had been compared to the corresponding negative control tissues.

The test item has no non-specific MTT-reducing or colouring potential, therefore no additional controls were necessary. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% (9.4%) after 60 min treatment but not below 50% (80.7%) after 3 min treatment. The controls confirmed the validity of the study. The mean OD_570nmof the two negative control tissues was 0.8 and ≤ 2.8 for each exposure period. The mean relative tissue viability (% negative control) of the positive control was < 15% (7.6%) after 60 min treatment. The coefficient of variation (CV) (in the range of 20 – 100% viability) of replicate tissues of all dose groups was 30% (5.0% - 8.1%).

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

In this study under the given conditions the test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% after 60 min treatment but not below 50% after 3 min treatment. The test item is therefore classified as “corrosive“ in accordance with a combination of optional UN GHS sub-categories 1B and 1C.

Executive summary:

In the present study the skin corrosivity potential of 4-Aminopyridine was analysed. Since corrosive chemicals are cytotoxic after a short time exposure to the stratum corneum of the epidermis the cytotoxic effects of the test item on EpiDerm , a reconstituted three-dimensional human epidermis 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 3 min and 60 min exposure period and compared to those of the concurrent negative controls. The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/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 test item per 300 µL Aqua dest.and per 90 µL isopropanol showed no colouring as compared to the solvent. Therefore NSC equaled 0%. The test item showed corrosive effects. The mean relative tissue viability (% negative control) was reduced below 15% (9.4%) after 60 min treatment but not below 50% (80.7%) after 3 min treatment.

Reference 2

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

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 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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

2.6

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

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 %).

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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

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

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%).

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%).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available