Hydrogen [29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with dodecylamine (1:1)

Endpoint summary

Description of key information

The substance does not show a skin corrosive potential in the EpiDerm™ skin corrosion test. This is consistent with in-vivo data of related substances.
The substance does not show an eye corrosive potential in a HET-CAM in vitro corrosion test, but it showed an irritant potential in the EpiOcular assay.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation / corrosion

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiDerm™ 200 kit

Details on test animals or test system and environmental conditions:

TEST SYSTEM:
- Source: MatTek Corporation, Ashland MA, USA

Three dimensional human epidermis model
The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Based an the results of an ECVAM (European Center tor Validation of Alternative Methods) funded prevalidation study, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human skin model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals. In the Draft OECD Guideline 431 it is suggested as model tor in vitro corrosivity testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37

Vehicle:

water

Controls:

other: historical control values

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL bulk volume of test material

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL doubly distilled water

Duration of treatment / exposure:

3 minutes at room temperature
1 hour in the incubator

Observation period:

No post-incubation period

Number of animals:

Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator) and test group (test material, negative control and positive control)

Details on study design:

EXPERIMENTAL PROCEDURE
Direct MTT reduction
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below.
Approximately 25 µL bulk volume added to 0.9 mL of the MTT solution. The mixture is incubated in the dark at about 37°C for 55 to 65 minutes. A negative control (50 µL of doubly distilled water) is tested concurrently.
lf the MTT solution color or in case of water-insoluble test substances the border to the water-phase turns blue / purple, the test substance is presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsity the test results.
In case that direct MTT reduction occurs one freeze-killed control tissue per exposure time is treated with, each, the test article and the negative control, in the same way as described in section "Basic procedure", additionally.

Basic procedure
On day of receipt EpiDerm™ tissues are kept in the refrigerator. At least 1 hour but not more than 1.5 hours before test substance application, tissues are transferred to 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. The preincubation medium is replaced with fresh medium immediately before application.
Two tissues per exposure time (3 minutes at room temperature or 1 hour in the incubator, as a rule) and test group (test material, negative control and positive control; 12 tissues per test) are used. If applicable , 1 killed tissue per exposure time is treated to control for direct MTT reduction.
A bulk volume of 25 µL of the test material is applied with a sharp spoon. Thereafter 25 µL doubly distilled water are added and homogeneously distributed together with the test substance.
Control tissues are concurrently applied with 50 µL of doubly distilled water (negative control, NO) or with 50 µL of 8n potassium hydroxide (positive control, PC) or test substance (killed tissue control, KC).
The tissues are washed with PBS to remove residual test material 3 minutes or 1 hour after start of treatment. Rinsed tissues are kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time are dosed and rinsed. The assay medium is then replaced by MTT solution and tissues are incubated for 3 hours.
After incubation, tissues are washed with PBS and the formazan produced by the tissues is extracted with isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts is determined spectrophotometrically. Blank values are established of 3 microtiter wells filled with Isopropanol for each microtiter plate.

Data evaluation
Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) together with the respective exposure time is used for evaluating whether or not a test material is corrosive.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent for each exposure time.

ACCEPTANCE CRITERIA

Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0.

Acceptance criteria for the positive control (PC)
Potassium hydroxide as 8.0 normal ready made solution is used as positive reference. A 3-minute treatment with 8.0 n KOH usually reveals a mean relative tissue viability of ~20%. An assay is acceptable if mean relative tissue viability of the 3 min positive control is ≤ 30%.

Assay acceptance criterion for tissue variability
For every treatment, except the killed controls, 2 tissues are treated in parallel. The inter-tissue variability is considered to be acceptable if the difference of the OD570 values of the two tissues is ≤ 0.3.

Assay acceptance criterion for killed controls (KC)
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35.

EVALUATION OF RESULTS
Corrosivity potential of the test materials is predicted from the mean relative tissue viabilities obtained after 3 min treatment compared to the negative control tissues concurrently treated with doubly distilled water. A chemical is considered as "corrosive", if the mean relative tissue viability after 3 min treatment with a test material is decreased below 50%. In addition, those materials with a viability of ≥ 50% after 3 min treatment are considered as "corrosive" if the mean relative tissue viability after 1 hour treatment with a test material is decreased below 20%.
Mean tissue viability
(% of negative control) Prediction
3 min: < 50 corrosive
3 min: ≥ 50 and 1 hour: < 20 corrosive
3 min: ≥ 50 and 1 hour: ≥ 20 non-corrosive

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Score:

93

Remarks on result:

other: corrosion, exposure: 3 min, mean OD570: 1.947

Irritation parameter:

other: viability [% of NC]

Basis:

mean

Score:

95

Remarks on result:

other: corrosion, exposure: 1 hour, mean OD570: 1.920

Irritant / corrosive response data:

Due to the ability of the test substance to reduce MTT directly, a KC (killed tissue control) was introduced. However, the result of the KC did not indicate an increased MTT reduction, therefore the KC was not used for viability calculation.
The value for inter tissue variability of the test substance for the exposure period of 1 hour is about 0.4 and therefore out of the acceptance range. Since the lower value still corresponds to a viability of 85%, this deviation is not considered to adversely affect the result of this study.
Based on the observed results and applying the evaluation criteria it was concluded, that the substance does not show a corrosive potential in the EpiDerm skin corrosivity test under the test conditions chosen.

Interpretation of results:

other: not corrosive

Remarks:

Criteria used for interpretation of results: EU

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:

weight of evidence

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, protocol internally validated for distinguishing non-irritants from irritants

Qualifier:

according to guideline

Guideline:

other: OECD draft testing guideline 492 (RECONSTRUCTED HUMAN CORNEA-LIKE EPITHELIUM (RHCE) TEST METHOD FOR IDENTIFYING CHEMICALS NOT REQUIRING CLASSIFICATION AND LABELLING FOR EYE IRRITATION OR SERIOUS EYE DAMAGE)

Principles of method if other than guideline:

Determination of eye damage in a reconstructed three dimensional human cornea model (EpiOcular™)

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

not applicable

The EpiOcularTM model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

To assess the ability of the test material to directly reduce MTT a pretest was performed.

Vehicle:

unchanged (no vehicle)

Controls:

other: yes (tissue incubations for positive and negative controls included)

Amount / concentration applied:

0.05 mL (33 mg)

Duration of treatment / exposure:

6 hours

Observation period (in vivo):

18h

Number of animals or in vitro replicates:

Two tissue samples were used per group.

Details on study design:

Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the testsubstance treated epidermal tissues is compared to that of negative control tissues. The
quotient of the values indicates the relative tissue viability.

Irritation parameter:

other: Viability (%)

Basis:

other: culture 1

Score:

33.7

Max. score:

100

Reversibility:

not specified

Irritation parameter:

other: Viability (%)

Basis:

other: culture 2

Score:

16.2

Max. score:

100

Reversibility:

not specified

Methyl acetate was used as positive control and deionised water as negative control and both gave the results to satisfy acceptance criteria.

Methyl acetate decreased the mean viability to 24% of control cultures.

Interpretation of results:

irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The mean viability was less than 60%, therefore the substance is considered to be irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION

The substance itself has been tested in an in-vitro study for skin corrosion according to OECD testing guideline 431 (BASF 2003). In this study, viability was not adversely effected for both the 3min and 60min exposure. This clearly shows absence of corrosive properties and is also indicative of a low irritation potential.

Specifically, in the GLP compliant in vitro EpiDerm™ test according to OECD guideline 431, the potential of the test substance to cause dermal corrosion was assessed by a single topical application of 25μL bulk volume (about 9 mg) of the test substance to a reconstructed three dimensional human epidermis model. Two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, each. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. Viability of the test substance treated tissues determined after an exposure period of 3 minutes was 93%. Viability of the test substance treated tissues determined after an exposure period of 1 hour was 95%. These results do not indicate a corrosive potential of the test substance. Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a corrosive potential in the EpiDerm™ skin corrosivity test under the test conditions chosen.

Profiling for skin irritation with the OECD QSAR Toolbox resulted in no alert for “inclusion” and in several alerts for exclusion (eg high molecular weight and high melting point). The molecular weight of > 540 g/mol is given as rule for “not classified with R38, R34 and R35” which are basicly equivalent to GHS irrancy categories 1 and 2.

 

The closest structural analoge is CAS 85650-96-0. It differs from the target substance in the length of the alkyl change of the amine (octadecane versus dodecane ) and in the presence of one unsaturated C=C bond in the alkylchain. Due to the longer alkyl chain it has higher molecular weight. Both substances are surface active and have very similar values for surface tension.  Considering the high molecular weight of greater than 500 g/mol, the slightly longer alkyl chain does not affect the irritating properties. Both substances share the same functional groups (sulfonic acids, alkylamine cations) that could convey irritating properties. Both substances are irritating to eyes, have surface activity and are of low acute oral toxicity.

The experimental data on this source substance is only valid with restriction as it was performed in the year 1980 prior to the introduction of GLP. The protocol differs from the OECD guideline no 404 in a longer exposure period (24h versus 4h), a shorter observation period (8days versus 14days) and the testing of both abraded and intact skin. It was performed according to the US Federal Register 38 , No. 187, § 1500.41 , p. 27029 of Sep. 27, 1973 (Draize test). In that study, erythema could initially not be scored because the blue colorant stained the application site. There were no edema and all findings were fully reversible within the observation period. Scores were clearly below the threshold for classification and labelling.

 

In supportive evidence, the data matrix lists further structural analogues which either have a higher degree of substitution (eg CAS 108300 -90 -9) or are sulfonic acids or its sodium salt. Some of them are not classified for irritation to eyes whereas others are highly irritating to eyes. None of them are classified for skin irritation based on valid experimental data.

It is referred to the attachement with supportive information (structures and data matrix) both in this Enspoint summary and in the chemical safety report. The current IUCLID template of an endpoint summary does not allow entering chemical structures.

Overall, it is considered acceptable to conclude in a weight-of-evidence approach that the substance does not need to be classified as a skin irritant.

EYE IRRITATION

The substance has been tested in two in-vitro studies which in combination allow classification and labelling for eye irritation.

In a non-GLP HET-CAM in vitro corrosion test, 3 chicken eggs were exposed for 5 minutes to the undiluted test substance. 300 seconds after exposure the effects observed after test substance removal by washing were graded.

Neither haemorrhagia nor coagulation were observed within the observation period of 300 seconds. Therefore, the substance is non-corrosive under the conditions of this test. Absence of corrosive properties is also indicated from the BfR irritation rules, specifically the high molecular weight and the high melting point.

To clarifiy the irritating properties, an in-vitro study for eye irritation (EpiOcular) was performed (BASF 2015). The study followed the draft of the OECD testing guideline 492 and GLP principles. In this study, the substance was identified as an eye irritant because treatement with the test substance reduced the mean viability to less than 60% (33 and 16% for each of the two treated cultures).

Therefore, the substance is considered to be irritating, but not highly irritating/corrosive to eyes.

Effects on eye irritation: irritating

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin irritation under Directive 67/548/EEC. Based on valid in-vitro studies, the substance is classified as an irritant to eyes (R36).

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin irritation under Regulation (EC) No. 1272/2008. It is however considered to be classified for eye irritation (H319).