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Description of key information

An initial in silico screening for skin sensitisation employing DEREK assessment did not give any alerts for skin sensitising properties of the substance. However, the phthalocyanine substructure could not be found in the negative prediction database and therefore the prediction of the substance to be a non-sensitiser should be interpreted with some reservations. The results of a solubility test indicated that substance could not be dissolved in any appropriate solvents to perform a valid DPRA assay. The solubility test also showed that the substance is not dissolvable in organic or aqueous solvents at workable concentrations for the KeratinoSensTM assay and U-SENSTM assay. Therefore, these studies are technically not feasible to perform. The DEREK result alone is insufficient to conclude on skin sensitising properties of the substance and thus an in vivo study was conducted to address the skin sensitising properties of substance.

In an in vivo local lymph node assay the substance was administered to groups of five female CBA/J mice. The substance was applied onto the ear at concentrations of 0, 1, 2 or 5% w/w on three consecutive days, following which all animals were injected with 3H-methyl thymidine and after five hours the lymph nodes were drained, excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The mean disintegrations/min /animal values for the experimental groups treated with substance concentrations 1, 2 and 5% were 918, 467 and 823, respectively. The mean DPM/animal value for the vehicle control group was 615 DPM.

The stimulation index values calculated for the substance concentrations 1, 2 and 5% were 1.5, 0.8 and 1.3, respectively.

Results showed no skin irritation in any of the animals. No mortality occurred and no clinical signs related to systemic toxicity were observed in the animals. Body weights and body weight gain of animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals in the treatment and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Therefore, the substance is considered as non-sensitising to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
other: Weight of evidence
Adequacy of study:
weight of evidence
Study period:
12th March 2019 - 12th March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Weight of evidence
Justification for type of information:
Weight of evidence
Principles of method if other than guideline:
As the substance is a mono-constituent and not a metal the following strategy was followed:
STEP 0: The starting point for the weight of evidence is the assessment whether new studies are required. Available information on the substance was used to evaluate whether:
- The substance is not a strong acid (pH≤ 2.0) or base (pH ≥ 11.5), known to be not corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature.
- No adequate existing human data, which provide evidence that the substance is a skin sensitizer are available.
- No data from existing studies on skin sensitization in laboratory animals, which provide sound conclusive evidence that the substance is a sensitizer or non-sensitizer, are available.

If no reliable data on solubility is available, a solubility test is performed to determine whether the substance dissolves sufficiently in a solvent, which is appropriate for each test mentioned below. In case the solubility test demonstrates solubility of the substance that meets the precondition limits for the in vitro tests, the following step-wise testing approach is followed:
STEP 1: DEREK assessment (overall skin sensitizing events), Direct Peptide Reactivity Assay (DPRA; molecular interaction with skin proteins) and KeratinoSensTM assay (inflammatory response in keratinocytes) are performed.
STEP 2: Depending on the outcome of the studies performed in STEP 1 and in absence of a definite conclusion on possible skin sensitization, the U-SENSTM assay is performed (key event: activation of dendritic cells).
STEP 3: Based on a weight of evidence of all available data on the substance related to skin sensitization, an argument is prepared to conclude on the classification for the substance or, if no conclusion can be drawn, to conclude on the performance of an in vivo skin sensitization study.
GLP compliance:
not specified
Type of study:
other: WOE
Specific details on test material used for the study:
CAS number: 2126827-44-7
Molecular weight: 1451.91
Purity/Composition: 99%
UVCB: No
Positive control results:
N/A
Run / experiment:
other: 1
Parameter:
other: Weight of evidence
Remarks:
Summary of DEREK and DPRA studies
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: Study cannot be used for classification
Remarks:
Value of '0' used to fulfill the completeness check of the dossier

No data were available that would preclude performance of the studies to determine the potential for skin sensitization. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment and a DPRA was initiated.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitization of the substance. However, the phthalocyanine substructure of the query structure was not found in the Lhasa skin sensitization negative prediction dataset (unclassified). 2,9,16,23-Tetrakis(2,5-di-tertbutyl-4-methoxyphenoxy)phthalocyanine is predicted to be not sensitizing to the skin, but this prediction should be considered with caution.

A solubility test with the substance was performed to determine an appropriate solvent for use in the DPRA. At a concentration of 100 mM, substance was not soluble in ACN, MQ, ACN:MQ (1:1, v/v), isopropanol, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), ethanol and methanol.

The solubility of substance was also assessed at lower concentrations in ethanol down to 10 mM at which substance still was not soluble. Since no suitable solvent was available, the DPRA could not be performed.

The KeratinoSensTM assay was not initiated as the information on solubility of the substance in several solvents indicated that no suitable solvent is available to perform this assay with.

Interpretation of results:
study cannot be used for classification
Conclusions:
According to the weight-of-evidence, an in silico approach employing DEREK NEXUS (6.0.1) did not yield any alerts for skin sensitisation. Further planned tests, DPRA and KeratinoSnens, U-SENS assays could not have been conducted due to the insolubility of the substance in the range of acceptable solvents.
Executive summary:

A DEREK assessment did not give any alerts for skin sensitizing properties of the substance. However, the phthalocyanine substructure could not be found in the negative prediction database and therefore the prediction of the substance to be a non-sensitizer should be interpreted with some reservations. The results of a solubility test indicated that substance could not be dissolved in any appropriate solvents to perform a valid DPRA with. The solubility test also showed that the substance is not dissolvable in organic or aqueous solvents at workable concentrations for the KeratinoSensTM assay and U-SENSTM assay. Therefore, these studies are technically not feasible to perform. The DEREK result alone is insufficient to conclude on skin sensitizing properties of the substance and thus additional information is required. In accordance with point 8.3.2 of Annex VII of the REACH Regulation, as in vitro/in chemico test methods are not applicable or adequate for classification and risk assessment it is scientifically justified to conduct an in vivo study to address the skin sensitizing properties of substance.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2nd April 2019 - 30th April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay.
Version / remarks:
July 2012
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020 (expiry date)
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light

Additional information
Test Facility test item number: 210026/A
Test item handling: Use amber glassware or wrap container in aluminum foil
CAS number: 2126827-44-7
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Husbandry
Housing
On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Makrolon MIII type; height 18 cm.) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22 to 23°C with an actual daily mean relative humidity of 41 to 45%. A 12 hour light/12 hour dark cycle was maintained. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Food
Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures.
The feed was analyzed by the supplier for nutritional components and environmental contaminants. Results of the analysis were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Municipal tap-water was freely available to each animal via water bottles.
Periodic analysis of the water was performed, and results of these analyses are on file at the Test Facility.
It is considered that there were no known contaminants in the water that would interfere with the objectives of the study.

Animal Enrichment
For psychological/environmental enrichment, animals were provided with paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home, MCORN 404, Datesand Ltd, USA), except when interrupted by study procedures/activities.

Veterinary Care
Veterinary care was available throughout the course of the study; however, no examinations or treatments were required.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 1, 2 and 5 % (w/w)
No. of animals per dose:
5
Details on study design:
Pre-screen Test
A pre-screen test was conducted in order to select the highest substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.
Two substance concentrations were tested; a 5% and 10% concentration. The highest concentration was the maximum concentration that could technically be applied.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

Main Study
Three groups of five animals were treated with one substance concentration per group. The highest concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the substance.

Excision of the Nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were euthanized by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.

Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 µm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4 ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Key result
Parameter:
SI
Value:
1
Variability:
Standard error of the mean ± 0.1
Test group / Remarks:
0
Remarks on result:
other: No indication of skin sensitization
Key result
Parameter:
SI
Value:
1.5
Variability:
Standard error of the mean ± 0.2
Test group / Remarks:
1 % w/w
Remarks on result:
other: No indication of skin sensitization
Key result
Parameter:
SI
Value:
0.8
Variability:
Standard error of the mean ± 0.1
Test group / Remarks:
2 % w/w
Remarks on result:
other: No indication of skin sensitization
Key result
Parameter:
SI
Value:
1.3
Variability:
Standard error of the mean ± 0.3
Test group / Remarks:
5 % w/w
Remarks on result:
other: No indication of skin sensitization
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with substance concentrations 1, 2 and 5% were 918, 467 and 823 DPM, respectively. The mean DPM/animal value for the vehicle control group was 615 DPM.

Skin Reactions / Irritation

No irritation was observed in any of the animals. Green substance remnants were present on the dorsal surface of the ears of all substance treated animals throughout the observation period, which did not hamper scoring of the skin reactions.

Systemic Toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. 

Green discoloration of the feces was noted for all substance treated animals between Days 3 and 5. In addition, green discoloration of the urine was noted for the animals treated at 5% on Day 3.

 Macroscopic Examination of the Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with substance concentrations 1, 2 and 5% were 918, 467 and 823 DPM, respectively. The mean DPM/animal value for the vehicle control group was 615 DPM. The SI values calculated for the substance concentrations 1, 2 and 5% were 1.5, 0.8 and 1.3, respectively.

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is considered to be non-sensitizing to skin based on a reliable in vivo skin sensitization study conducted according to OECD TG 429.
Executive summary:

In an in vivo local lymph node assay conducted according to the OECD TG 429 the substance was administered to groups of five female CBA/J mice. The highest concentration of the substance was chosen from a pre-screen test which showed no sensitizing potential at the highest dose of 10 % w/w of the substance. The substance was applied onto the ear at concentrations of 0, 1, 2 or 5% w/w on three consecutive days. Three days after the last exposure all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed.

The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group. Mean DPM/animal values for the experimental groups treated with substance concentrations 1, 2 and 5% were 918, 467 and 823 DPM, respectively. The mean DPM/animal value for the vehicle control group was 615 DPM. The SI values calculated for the substance concentrations 1, 2 and 5% were 1.5, 0.8 and 1.3, respectively.

Results showed no skin irritation in any of the animals. No mortality ocurred and no clinical signs related to systemic toxicity were observed in the animals. Body weights and body weight gain of animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals in the treatment and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted.

Therefore, the substance is considered as non-sensitising to skin.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the findings of a reliable in vivo skin sensitisation study conducted on the substance, classification of the substance is not justified.