Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Endpoint summary

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:

January 14 - January 21, 2013

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)

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): 36.8 - 37.4
- Humidity (%): 79 - 96

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: 11.7 to 14.2 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.: 13-EKIN-001).This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates 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:

% tissue viability

Value:

77

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Percentage of control. Time point: 15 minutes. Remarks: Negative control = 100%; Positive control = 5%.

Other effects / acceptance of results:

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 7 compared to the negative control tissues was 77%. Since the mean relative tissue viability for Amiloride Compound 7 was above 50% Amiloride Compound 7 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:

GHS criteria not met

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 7 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 Standard Model), the influence of Amiloride Compound 7 on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (11.7 to 14.2 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 5% whereas the test substance showed cell viability of 77%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that Amiloride Compound 7 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, other

Remarks:

other: expert judgement

Type of information:

other: expert judgement

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Principles of method if other than guideline:

Combination of BCOP assay result for the substance in question and abbeviated results froman in vivo study conducted pre-guideline and GLP.

Species:

rabbit

Vehicle:

physiological saline

Amount / concentration applied:

0.1 ml at 10 %

Observation period (in vivo):

96 hours

Number of animals or in vitro replicates:

4

Irritation parameter:

other: qualitative

Basis:

mean

Time point:

other: 96 hrs.

Score:

ca. 0

Reversibility:

fully reversible within: 96 hrs.

Remarks on result:

not determinable because of methodological limitations

Irritant / corrosive response data:

Amiloride 7 BCOP in vitro score 1.4- not a severe eye irritant.

In vivo: dated 15 March 1965
Species: rabbit
No tested: 4
Instillation:  0.1ml at 10% concentration in saline
Results: Produced apparent "stinging, slight injection of the conjunctivae, chemosis and discharge persisting through 24 hours. The eyes were normal whenexamined at 96 hours.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

The results from the BCOP assay conducted would indicate that the substance is not classified for serious eye damage, in addition the minimal in vitro score would indicate that the substance would have negligible effects on the ocular tissue in vivo. The results from the in vivo study, while not sufficient in themselves to make a conclusive decision on classification, in combination with the BCOP results indicate that the substance is none irritant.

Executive summary:

A weight of evidence determination has been carried out to assess the eye irritation potential of Amiloride 7, based upon the results of the in vitro BCOP assay and historical in vivo data. The results of the BCOP assay are overwhelmingly negative, while the observations in 4 rabbits with no corneal or iris involvement and only minimal conjunctival involvement which resolve by 96 hours indicate that the substance is not a serious eye irritant and would not fulfill the EU CLP critieria for classification.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:

A modern study in accordance with EU Method B46, conducted to GLP

Justification for selection of eye irritation endpoint:

A weight of evidence determination drawing information from a modern GLP study conducted to an EU Method and historical in vivo studies, to derive a robust classification.

Justification for classification or non-classification

Skin Irritation:

Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 5% whereas the test substance showed cell viability of 77%. Since the mean relative tissue viability after exposure to the test substance was above 50%, it can be concluded that Amiloride Compound 7 is not irritating in the in vitro skin irritation test. 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 7 is not irritating in the in vitro skin irritation test.

Eye Irritation:

A weight of evidence determination has been carried out to assess the eye irritation potential of Amiloride 7, based upon the results of the in vitro BCOP assay and historical in vivo data. The results of the BCOP assay are overwhelmingly negative, while the observations in 4 rabbits with no corneal or iris involvement and only minimal conjunctival involvement which resolve by 96 hours indicate that the substance is not a serious eye irritant and would not fulfill the EU CLP critieria for classification.