Registration Dossier

3-MPA has shown skin corrosivity in vitro and in vivo. It caused irreversible cornea damage in one rabbit. Rats exposed to 3-MPA aerosol exhibited irritation of the respiratory tract only at high dosages.

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-19 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): At the end of the exposure period each EpiDerm tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS).
- Time after start of exposure: 3 min, 60 min

SCORING SYSTEM: Data are presented in the form of % viability (MTT conversion relative to negative controls) for each of the two exposure times.
Classification of corrosivity potential was based on relative viabilities for both exposure times according to the Table 1 (Material & Methods).

Amount/concentration applied:

50 µL, undiluted

Duration of treatment / exposure:

3 min, 60 min

Duration of post-treatment incubation (if applicable):

3 h

Number of replicates:

duplicate tissues were used per exposure time

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min exposure

Value:

5.26

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min exposure

Value:

5.24

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

see Table 2

The relative mean viability was 5.26% after 3 minutes exposure, and 5.24% after 60 minutes exposure. The relative mean viability of the test material-treated tissues was <10% after 3 minutes exposure.

Table 2: Mean OD₅₄₀Values and Mean % Viabilities for the Negative Control, Positive Control Material and Test Material

Material	Exposure Time	Mean OD₅₄₀¹	Mean % Viability¹
Negative control distilled water	3 minute	1.977	100%
Negative control distilled water	60 minute	1.986	100%
Positive Control Material	3 minute	0.109	5.51²
Positive Control Material	60 minute	0.100	5.04³
Test Material	3 minute	0.104	5.26²
Test Material	60 minute	0.104	5.24³

¹= Mean of 2 EpiDerm tissues tested in duplicate

²= Mean viability expressed as a percentage of the mean viability of the 3 minute negative control tissues

³= Mean viability expressed as a percentage of the mean viability of the 60 minute negative control tissues

Interpretation of results:

Category 1B (corrosive) based on GHS criteria

Conclusions:

The test material is considered to have the potential to be corrosive in vivo.
GHS classification: Danger, Skin Corrosive. Category 1B; H314: Causes severe skin burns and eye damage.

Executive summary:

The study was performed to assess the corrosivity potential of the test material using the EpiDerm Skin Model (MatTek, Ashland, MA, USA). The test is based on the assumption that corrosivity potential is related to toxicity to the EpiDerm tissue. The study was validated by the inclusion of a positive control material, 8.0 n Potassium Hydroxide and a negative control material, sterile distilled water.

The experimental design of the study consists of a test for Direct Reduction of MTT by the test material, followed by the main study.
For the main study, duplicate EpiDerm tissues were treated with 50 μl of test material and exposed for 3 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure times.
Negative control-treated tissues (50 μl sterile, distilled water), and positive control-treated tissues (8.0 n Potassium Hydroxide), were also exposed for 3 minutes and 60 minutes. Duplicate EpiDerm tissues were used for the above.

At the end of the exposure period each EpiDerm tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS) according to the modified rinsing procedure (Appendix 2) and placed into a ‘holding plate’, until all of the tissues had been treated and rinsed. They were then transferred to an MTT ‘loading plate’, and incubated at 37°C for 3 hours in a humidified atmosphere of 5% CO2 in air. At the end of this time, each EpiDerm tissue was blotted dry and placed into an MTT ‘extraction plate’ in order to extract all of the reduced MTT from the tissues.
At the end of the extraction period, the extracted MTT solution was mixed for each EpiDerm tissue and 3 x 200 μl samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured with the Anthos 2001 microplate reader. Data are presented in the form of % viability (MTT conversion relative to negative controls) for each of the two exposure times.

The ability of the test material to directly reduce MTT in the direct MTT reduction test proved inconclusive. There was a possibility that if the test material could not be totally rinsed off the EpiDerm tissues, that any residual test material present on the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore a corrective procedure using a “freeze killed” control EpiDerm tissue, also treated with the test material, was necessary to quantify this possibility.
The relative mean viability was 5.26% after 3 minutes exposure, and 5.24% after 60 minutes exposure.
The relative mean viability of the test material-treated tissues was <10% after 3 minutes exposure. The test material is considered to have the potential to be corrosive in-vivo.

Endpoint conclusion:

adverse effect observed (corrosive)

Endpoint conclusion:

adverse effect observed (irreversible damage)

Endpoint conclusion:

adverse effect observed (irritating)

Skin irritation

The study was performed to assess the corrosivity potential of the test material using the EpiDerm Skin Model (MatTek, Ashland, MA, USA). The test is based on the assumption that corrosivity potential is related to toxicity to the EpiDerm tissue. The study was validated by the inclusion of a positive control material, 8.0 n Potassium Hydroxide and a negative control material, sterile distilled water.

The experimental design of the study consists of a test for Direct Reduction of MTT by the test material, followed by the main study.
For the main study, duplicate EpiDerm tissues were treated with 50 μl of test material and exposed for 3 minutes and 60 minutes. The tissues were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for the appropriate exposure times.
Negative control-treated tissues (50 μl sterile, distilled water), and positive control-treated tissues (8.0 n Potassium Hydroxide), were also exposed for 3 minutes and 60 minutes. Duplicate EpiDerm tissues were used for the above.

At the end of the exposure period each EpiDerm tissue was rinsed using Dulbecco’s phosphate buffered saline (DPBS) according to the modified rinsing procedure (Appendix 2) and placed into a ‘holding plate’, until all of the tissues had been treated and rinsed. They were then transferred to an MTT ‘loading plate’, and incubated at 37°C for 3 hours in a humidified atmosphere of 5% CO2 in air. At the end of this time, each EpiDerm tissue was blotted dry and placed into an MTT ‘extraction plate’ in order to extract all of the reduced MTT from the tissues.
At the end of the extraction period, the extracted MTT solution was mixed for each EpiDerm tissue and 3 x 200 μl samples for each tissue were transferred to the appropriate wells of a 96 well plate. The absorbency at 540nm (OD540) of each well was measured with the Anthos 2001 microplate reader. Data are presented in the form of % viability (MTT conversion relative to negative controls) for each of the two exposure times.

The ability of the test material to directly reduce MTT in the direct MTT reduction test proved inconclusive. There was a possibility that if the test material could not be totally rinsed off the EpiDerm tissues, that any residual test material present on the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore a corrective procedure using a “freeze killed” control EpiDerm tissue, also treated with the test material, was necessary to quantify this possibility.
The relative mean viability was 5.26% after 3 minutes exposure, and 5.24% after 60 minutes exposure.
The relative mean viability of the test material-treated tissues was <10% after 3 minutes exposure. The test material is considered to have the potential to be corrosive in-vivo.

Eye irritation

The study does not need to be conducted because the substance is classified as skin corrosion, leading to classification as serious eye damage (Category 1).

3-MPA has to be classified as strongly corrosive, based on in vivo and in vitro results.

GHS classification: Danger, Skin Corrosive. Category 1B; H314: Causes severe skin burns and eye damage.