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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2018-02-06 to 2018-04-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: method B.51 of the council regulation No.640/2012 adopted 06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

1
Chemical structure
Reference substance name:
Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.
EC Number:
309-627-7
EC Name:
Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.
Cas Number:
100545-46-8
Molecular formula:
C32H16-τN8Cu.(SO3)m.(SO3H)p.(C9H19N2SO2)O.(C9H20N2SO2)n
IUPAC Name:
Copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, [[3-(cyclohexylamino)propyl]amino]sulfonyl derivs.
Test material form:
solid: particulate/powder
Specific details on test material used for the study:

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941) Le Genest Saint Isle
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 18.8 - 22.9 g

- Housing:
* Cages: solid-floor polypropylene cages with softwood woodflakes.
* Individually: to avoid ingestion of test item or licking of the ear
* Environmental enrichment items are provided to animals and considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

- Diet : ad libitum
- Water : ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19 - 25 °C
- Humidity 30- 70%
- Air changes : at least 10 times per hour
- Photoperiod: 12 hrs dark / 12 hrs light


Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Remarks:
This vehicle was chosen as it produced the most suitable formulation at the required concentration.
Concentration:
10, 25 , 40% of test item in dimethylformamide (DMF)
No. of animals per dose:
1 (preliminary study at 60%)
2 (prelimnary study at 40% and 25%)
4 (main study)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was a 60% (w/w) dilution in DMF

RANGE-FINDING STUDY:

- Compound solubility: The highest test item concentration, which could be technically used, was a 60% (w/w) dilution in DMF
- Concentrations tested : 60% test item diluted in DMF, 40%, and 25% test item diluted in DMF.
- Irritation: no signs of erythema
- Systemic toxicity: toxicity was not observed
- Ear thickness measurements: the ear weight did not show toxicity, but it was increased when compared to the negative control group
- Weight Lymph nodes: no signs of toxicity


PRELIMINARY STUDY:



- Irritation:

* At 60% of test item in DMF, there was a significant ear thickness increase, with no sign of erythema
* at 25, and 40% of test item in DMF, no signs toxicity were observed.

- Systemic toxicity: no systemic toxicity

- Ear thickness measurements:
* at 60% test item diluted in DMF :
Day 1: 0.21 mm
Day 6: 0.42 mm

* at 40% test item diluted in DMF
Day 1: 0.19 mm
Day 6: 0.22 mm

* at 25% test item diluted in DMF
Day 1: 0.22 mm
Day 6: 0.22 mm


MAIN STUDY : As a result of the preliminary test, dose levels of 10%, 25% and 40% were selectedin the main study. Vehicle control groups were included in the main study.



ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU incoroporation measured in a photometer
- Criteria used to consider a positive response: The proliferative response of the lymph node cells is expressed as the mean S.I. The S.I. is derived by dividing the mean BrdU labeling index/mouse within each treated group by the mean BrdU labeling index for the vehicle group.
A test item is regarded as a sensitizer in the LLNA if :
Exposure to at least one concentration of the test item resulted to a stimulation index value equal or greater than 1.6 compared to the control values
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:

Test item preparation : The solutions of 10, 25 and 40% of test item in DMF are freshly prepared.
Test item Administration:
Daily topical application for three consecutives days (D1, D2 and D3) of 25µL of the appropriate test item/solvent solutions (10%, 25% or 40% test item in DMF and DMF), applied to the the dorsal surface of each ear
Two days after the third application on Day 5 (D5): ntraperitoneal injection of 0.5 mL (5mg/per mouse/injection) of BrdU solution (10 mg/mL) .
One day after BrdU injection (D6), animals were euthanized and the draining lymph nodes from each mouse was excised.
From each mouse, a single cell suspension of lymph node cells (LNC) was prepapred by gentle mechanical disaggregation through a 70 µm nylon mesh. The lymph node cells were re-suspended in 15 mL DPBS (Ca2+ / Mg2+ - free). into a well of a multi-well 6. The optimized volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2.
100 μL of the LNC suspension was added to the wells of a flat-bottom microplate at least in triplicate.
After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 μL of 1 M H2SO4 was added in each well, then shaken for one minute. The absorbance was measured at 450 nm with a reference wavelength of 690 nm.


Clinical observations and mortality : daily

Bodyweight :
- on day 1 prior dosing
- on day 6 prior termiantion

Ear thickness measurements and recording for local reactions: via a micrometer
- Prior dosing : day 1 and day 3
- After sacrifice : day 6

On day 6 : punch biopsies of 8mm in diameter of the apical areas of the two ears were prepared and weighed to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed




Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not provided.

Results and discussion

Positive control results:
In the positive control, the Stimulation index was 1.85

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
ca. 1.33
Test group / Remarks:
10% of test item
Key result
Parameter:
SI
Value:
ca. 1.22
Test group / Remarks:
25% of test item
Key result
Parameter:
SI
Value:
ca. 1.35
Test group / Remarks:
40% of test item
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION

Stimulation index values :
* 1.33 at 10%
* 1.22 at 25%
* 1.35 at 40%

EC1.6 CALCULATION
As SI <1.6, No EC 1.6 was determined

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity during the test

BODY WEIGHTS
Body weight changes in test animals between day 1 and day 6 were comparable to control animals

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results obtained, under these experimental conditions, enable to conclude that the test item does not have to be classified as a skin sensitizer, in accordance with the Regulation EC No. No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required.
Executive summary:

The test was performed to assess the skin sensitisation potential of the test item in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

The basic principle underlying the LLNA:BrdU is that sensitizers induce proliferation of lymphocytes in the lymph nodes draining the site of test item application.

Three groups of four animals were treated for three consecutive days (D1, D2, D3) with 50 μL (25 μL per ear) of the test item diluted at concentrations of 10%, 25% and 40% in N,N-dimethylformamide (DMF). A further group of four animals was treated with DMF.

On D5, 0.5 mL of BrdU solution (10 mg/mL) was injected by the intraperitoneal route.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. The experimental protocol was established according to the O.E.C.D. Guideline No. 442-B adopted 22 July 2010 and the test method B.51 of the council regulation No.640/2012 adopted 06 July 2012.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.

Blue coloration not preventing erythema quotation was noted in all animals treated at 10%, 25% and 40% between days 2 and 6.

No increase in ear thickness was noted in animals treated at 10%, 25% and 40%.

An increase in ear weight (+6.3%, +3.5% and +16.8%) was noted in animals treated at 10%, 25% and 40%, respectively.

Therefore, the test item has to be considered as not excessively irritant at these concentrations.

The Stimulation Index (SI) calculated by individual approach was 1.33, 1.22 and 1.35 for the treated groups at 10%, 25% and 40%, respectively.

Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6.

The results obtained, under these experimental conditions, enable to conclude that the test item does not have to be classified as a skin sensitizer, in accordance with the Regulation EC No. No. 1272/2008 on classification, labelling and packaging of substances and mixtures.

No signal word or hazard statement is required.