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EC number: 240-387-5 | CAS number: 16298-03-6
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Description of key information
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- 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.
Reference
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- 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
- 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.
Reference
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
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.
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