Methyl 3-aminopyrazinecarboxylate

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:

14-Jan-2013 to 21 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EU method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 439: In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The solid test substance (12.2 to 13.2 mg) was applied directly on top of the skin tissue. Amiloride Compound 6 was spread to match the size of the tissue.

NEGATIVE CONTOL:
- Amount(s) applied (volume or weight with unit): 25 µl Phosphate buffered saline

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 25 µl
- Concentration (if solution): 5% (aq) Sodium dodecyl sulphate

Duration of treatment / exposure:

Exposure:15 minutes
Post incubation period: 42 hours

Details on study design:

TEST SITE
- Area of exposure: human epidermis model
- % coverage: 0.38 cm2

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

POST INCUBATION PERIOD
- 42 hours

SCORING SYSTEM:
- After a 42 hour incubation period, determination of the cytotoxic (irritancy) effect was performed. 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: percentage viability

Value:

100

Remarks on result:

other: ercentage of control. Time point: 15 minutes.

Interpretation of results:

GHS criteria not met

Conclusions:

Amiloride Compound 6 is non-irritant in the in vitro skin irritation test

Executive summary:

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 6 compared to the negative control tissues was 100%. Since the mean relative tissue viability for Amiloride Compound 6 was above 50% after 15 minutes treatment Amiloride Compound 6 is considered to be non-irritant.

 

The positive control had a mean cell viability of 5% after 15 minutes exposure. The absolute mean OD₅₇₀(optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation

Remarks:

other: read-across/weight of evidence

Type of information:

other: expert judgement

Adequacy of study:

disregarded due to major methodological deficiencies

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Part of a weight of evidence: No experimental details are available.

Principles of method if other than guideline:

The eye irritation potential of the substance has been estimated from the weight of evidence presented by the results of BCOP assays conducted on the Amiloride series (intermediates 5, 6 and 7) in conjunction with the calculated pKa values for the carboxylic acid group on the respective Amiloride species and the results from an in vivo study conducted with Amiloride 7. In addition the measured partition co-efficients and surface activity of the three substances was been taken into account.

GLP compliance:

not specified

Predicted pKA and isoelectric point values for the 2 -carboxylic acid group (www.chemicalize.org/)

Amiloride 5; CAS 5424 -01 -1; 3-aminopyrazine-2-carboxylic acid:       pKa = 3.97; isoelectric point pI= 2.35.

Amiloride 6; CAS 16298 -03 -6; methyl 3 -aminopyrazine-2 -carboxylate: pKa = no ionization; isoelectric point pI= 8.63.

Amiloride 7; CAS 1458 -18 -0; methyl 5,6-dichloro-3 -aminopyrazine-2 -carboxylate: pKa = no ionisation; isoelectric point pI= 6.68

Measured Log P values:

Amiloride 5;       <=-3.1

Amiloride 6;       0.2

Amiloride 7;       2.2

Measured Surface Activity mN/m :

Amiloride 5;       73.6              negative

Amiloride 6;      73.3              negative

Amiloride 7;       74.7              negative

Interpretation of results:

GHS criteria not met

Conclusions:

The relative surface activity of the three related substances has been considered as a possible mechanism for induction of the severe eye irritation predicted by the BCOP assay conducted on Amiloride 5. The surface activity measurements are all similar and considered negative. The measured activities for the three substances indicates that the irritation predicted for Amiloride 5 is not mediated by surface activity.

Examination of the calculated pKa values for the Amiloride series would indicate that the severe irritation predicted by the BCOP assay for Amiloride 5 is a function of the ionised species present at physiological pH; there is a good correlation between the ionisation state of the molecules at pH 7 and the BCOP scores. The isoelectric points for Amiloride 6 and Amiloride 7 have been calculated to be within approx. 1 pH unit of physiological norm (pH7.4), therefore the majority of the species present will be in a non-ionised form, however Amiloride 5 will be present in its ionised form. It is proposed that severe eyedamage/seroius eye irritaiton will only be mediated by the ionised species of this series of substances.

The logP for Amiloride 5 is a function of the ionised species at pH7, the increased lipophilicity of Am6 is related to the ester bond formation.The logP values indicate that the relative degree of uptake into the ocular tissues for the series would follow the following pattern: Am7>Am6>Am5.
Given that minimal ocular effects are seen in vivo from the non-ionised Am7, it can be expected that a similar degree of damage would result from Am6.

The proposed classification for Amiloride 6 on the basis of expert judgement is not irritant.

Executive summary:

A weight of evidence determination has been carried out to assign a classification for serious eye irritation/severe eye damage.

The determination examined the following physical parameters for the related series of substances Amiloride 5, 6 and 7: surface activity, log P and calculated pKa and isoelectric points in relation to the effects seen in BCOP assays conducted on all three substances. Additionally, the in vivo effects observed for Amiloride 7 were taken into account when determining the eye irritation potential of Amiloride 6.

Surface activity can be discounted as a mechanism for the damage observed in the Amiloride 5 BCOP assay, since all the substances have a similar negative activity.

The calculated pKa and isoelectric point for Amiloride 5 indicates that it is present as the ionised carboxy species at physiological pH, while neither Amiloride 6 nor 7 are ionised at that pH. It is postulated that the positive result seen in the BCOP assay for Amiloride 5 is mediated by the ionised carboxylate group and that the negative effects seen for Amiloride 6 and 7 can be attributed to the lack of ionised species at physiological pH.

Finally, the lack of classifiable results seen in an in vivo study for Amiloride 7 can be read-across to Amiloride 6, on the basis of similar physical values for surface activity, log P, and calculated pKa and isoelectric point in conjunction with the negative BCOP assays seen for both substances.

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:

This study was performed according to Method B46 and in compliance with GLP. It is a modern, reliable study

Justification for selection of eye irritation endpoint:

The weight of evidence determination was based upon the physical values for log P, surface activity, pKa and isoeelctric point to provide a mechanistic interpretation for the effects seen in vitro BCOP for the three substances considered and allowed the in vivo results seen for Amiloride 7 to be read-across to Amiloride 6.

Justification for classification or non-classification

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 6 compared to the negative control tissues was 100%. Since the mean relative tissue viability for Amiloride Compound 6 was above 50% after 15 minutes treatment Amiloride Compound 6 is considered to be non-irritant.

 

The lack of classifiable results seen in an in vivo study for Amiloride 7 can be read-across to Amiloride 6, on the basis of similar physical values for surface activity, log P, and calculated pKa and isoelectric point in conjunction with the negative BCOP assays seen for both substances.