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Description of key information

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 17, 2012 - December 24, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2010)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2009)
Deviations:
no
GLP compliance:
yes
Species:
human
Details on test animals or test system and environmental conditions:
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9 - 37.4
- Humidity (%): 69 - 95
Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amounts applied: 10.8 to 12.1 mg, 5 μl Milli-Q water to moisten skin.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate
Duration of treatment / exposure:
15 minutes
Details on study design:
TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Lot no.: 12-EKIN-047). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days , which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.
Irritation / corrosion parameter:
other: other: tissue viability
Value:
110
Remarks on result:
other:
Remarks:
Basis: other: percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 31%. (migrated information)
Irritant / corrosive response data:
Skin irritation is expressed as the remaining cell viability after exposure to the test substance. The relative mean tissue viability obtained after 15 minutes treatment with Amiloride Compound 5 compared to the negative control tissues was 110%. Since the mean relative tissue viability for Amiloride Compound 5 was above 50% after 15 minutes treatment Amiloride Compound 5 is considered to be non-irritant.

The test substance was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because no colour change was observed it was concluded that the test substance did not interact with MTT.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
It is concluded that this test is valid and that Amiloride Compound 5 is not irritating in the in vitro skin irritation test.
Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Small Model), the influence of Amiloride Compound 5 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm2 cultured skin (10.8 to 12.1 mg, in presence of 5 μl Milli-Q water). After 15 minutes, the substance was removed and cells were cultured for 42 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 110%. Since the mean relative tissue viability after exposure to Amiloride Compound 5 was above 50%, it can be concluded that Amiloride Compound 5 is not irritating in the in vitro skin irritation test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

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
Study period:
December 10 - December 11, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 437 “Bovine corneal opacity and permeability (BCOP) test method for identifying ocular corrosives and severe irritants”
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EU Method B.47 “Bovine corneal opacity and permeability method for identifying ocular corrosives and severe irritants
Deviations:
no
GLP compliance:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 316 to 396 mg per cornea (undiluted)
Since no workable suspension of the test substance in physiological saline could be obtained, the test substance was used as such and added as such on top of the corneas.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole in physiological saline


Duration of treatment / exposure:
240 minutes (4 hours)
Details on study design:
TEST SITE
- Isolated bovine cornea

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: 240 minutes

SCORING SYSTEM:
- After exposure the cornea is thoroughly rinsed to remove the test substance followed by immediate opacity measurement and permeability evaluation of the cornea.

- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).


TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

DATA EVALUATION:
A test substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
146
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
111
Remarks on result:
positive indication of irritation

- The corneas treated with the substance showed opacity values ranging from 136 to 158 and permeability values ranging from -0.006 to 0.235

Interpretation of results:
Category 1 (irreversible effects on the eye)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Amiloride Compound 5 is corrosive in the Bovine Corneal Opacity and Permeability test.

Executive summary:

The negative control responses of the opacity and permeability values were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 111 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Amiloride Compound 5 induced ocular irritation through the opacity endpoint only, resulting in a mean in vitro irritancy score of 146 after 240 minutes of treatment.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Substance was highly irritating in a BCOP eye study and is therefore classified as eye damage 1.

Effects on eye irritation: highly irritating

Justification for classification or non-classification