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Diss Factsheets

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
experimental part performed in period. 28. 8. 2013 - 15. 10. 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out according to required test method in accordance with internationally valid GLP principles.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Copper phtalocyanine monosulfonated and disulfonated
EC Number:
Molecular formula:
C32H15N8Cu(HSO3)n where n=1.3-1.6
Copper phtalocyanine monosulfonated and disulfonated
Constituent 2
Reference substance name:
Copper phtalocyanine mono - and disulphonated
Copper phtalocyanine mono - and disulphonated
Constituent 3
Reference substance name:
Hysperse 12
Hysperse 12
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Copper phtalocyanine mono- and disulphonated
- Molecular formula: C32H15N8Cu(SO3H)1.4x2.5H2O
- Molecular weight:733.3
- Substance type: technical product
- Impurities: 0.5 % (w/w) water soluble salts
- Physical state:powder
- Lot/batch No.: 01
- Stability under test conditions: stable
- Storage condition of test material: The substance was stored in closed vessel in dry room at the temperature bellow 25ºC in dark.

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Breeding farm VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760118
- Age at study initiation: 8 to 10 weeks
- Weight at study initiation: 17.43 - 19.33 g
- Housing: group-wise five in macrolon cages with sterilized softwood shavings
- Diet: Pelleted standard diet for experimental animals ad libitum
- Water: Drinking tap water ad libitum.
- Acclimation period: At least 5 days

- Temperature: Room temperature 19.9 - 25.0 ºC, permanently monitored
- Humidity: Relative humidity 38.4 – 61.8 %, permanently monitored
- Photoperiod: 12 hours light/dark cycle

Study design: in vivo (LLNA)

other: DAE 433 - mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol
50% (v/v)
5% (v/v)
0.5% (v/v)

No. of animals per dose:
Pilot experiment: 3 females
Exposed groups: 15 females (5 animals in 3 groups)
Positive control group: 5 females
Negative control group: 5 females

Details on study design:
The three concentrations, 50%, 5 % and 0.5 % were administered to three animals to assess of systemic toxicity. The route of administration was the same as in the main study. During the pilot experiment no test substance related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected.
The test substance caused marked colouration of the skin on dorsum of both ears in the animals at the highest dose level, so assessment of erythema and other skin reactions could not be evaluated, but no clinical symptoms of systemic toxicity were observed. In othed animals no ertythema and skin reaction were observed. The thickness of ears in all animals during pilot experiment was not influenced by the treatment.
During the pathological examination no pathological changes was found.

The principle of the Local Lymph Node Assay (LLNA) is that sensitizers induce a primary proliferation of lymphocytes in the lymph node draining the site of chemicals application. The proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective, quantitative measurement of sensitisation. The LLNA allowes to access this proliferation as a dose-response relationship in which the proliferation in test groups is compared to that in vehicle treated controls, termed the Stimulation Index. The method is based on the use of radioactive labelling (3H-methyl thymidine) to measure cell proliferation.

Evaluation of results
Stimulation index (SI)
The SI is obtained by dividing the pooled radioactive incorporation for each treatment group by the incorporation of the pooled vehicle control group; this yields a mean SI.
The response towards the test substance is considered positive, if the stimulation index (SI) is ≥ 3, and the response increases in dose-related manner (dose-response relationship).
The response is considered negative, if the stimulation index (SI) is < 3 without the dose–response relationship.
The response is considered ambiguous if the stimulation index (SI) is < 3, but the response increases in dose-related manner (dose–response relationship), and eventually statistical significance is observed.
Validity criteria:
The test is considered valid, if the positive control substance DNCB (dinitrochlorobenzene) produces a positive LLNA response, and if the stimulation index SI is ≥ 3 over the negative control group.
Ear weight – irritation effect
If after treatment with the test substance a statistically significant increase of ear weight together with clear concentration dependence of the effect is recorded, the inflammatory effect is considered as irritation induced by the test substance.
A positive result in cell proliferation reveals that the test substance could be a contact allergen, but an irritation effect of test substance (increased ear weight) does not rule out the possibility that it can be a false positive result.

All suspensions were prepared by mixing an appropriate amount of the substance and the vehicle to obtain the application form in concentration of 50%, 5% or 0.5% (v/v). The suspensions were prepared before the start of application by mixing on magnetic stirrer.
The volume of the application form was constant at all groups of animals - 25 μL of the appropriate dilution to the dorsum of each ear once a day morning for 3 consecutive days. The application was performed very slowly by micropipette. The sixth day injection of 250 μL of phosphate-buffered saline (PBS) containing 7.35 x10e5 Bq of 3H-methyl thymidine into all test and control mice via the tail vein.
Five hours later, the animals were killed.

Health and mortality: at least twice daily during the dosing period
Clinical observations: at least twice daily during the dosing period
Body weight: on the first day of treatment, before dosing, and on the day of necropsy before application of the radionuclide

Ears weights: Immediately after scheduled humane killing of animals, the both ears were cut off and circular pieces from the apical area of each ear with a diameter of 8 mm were excised and weighed together.
Incorporation of 3H-methyl thymidine: The tissue of both of the lymph nodes was prepared by gentle mechanical disaggregation through 100 mm-mesh nylon gauze with concomitant pooling in 1 mL PSB (Phosphate Buffered Saline). The pairs of lymph nodes was analysed separately for each animal per group. Cells were washed twice with an excess of PBS and precipitated with 5% trichloroacetic acid (TCA) at 4 oC for 18h. The pellets were re-suspended in 1 mL TCA and transferred to scintillation vials containing 10 mL of scintillation fluid. Incorporation of 3H-methyl thymidine were was measured by β-scintillation counting (Beckmann LS 6500) as disintegrations per minute (DPM).

Mean values and standard deviations of ears weight and incorporation of 3H-methyl thymidine were computed for the test substance groups and for the positive as well as the vehicle control group. Stimulation index (for incorporation of 3H-methyl thymidine) was calculated by dividing mean values from exposed groups and the positive control group by the corresponding mean value of the vehicle control group. The index for the vehicle control
group was set at 1 by definition.

Positive control substance(s):
other: Dinitrochlorbenzene, 0.5% (w/v) solution. The vehicle and dosage volume were the same as in treated groups.
For statistical calculations the software Statgraphic ® Centurion (version XV, USA) was used. Statistical evaluation of measured parameters was performed at first by applying the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test, and then the non-parametric two-group
Mann-Whitney rank test (probability level 0.05) for all two-group comparisons.

Results and discussion

Positive control results:
In the positive control group, the SI was ≥ 3 (11.55) – test LLNA was efficient.

Irritating effect:
In the positive control group, the weight of ear target was statistically significantly increased as compared to the negative control group – the test design used is efficient in the detection of irritation effect.

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: See Table bellow
other: disintegrations per minute (DPM)
Remarks on result:
other: See Table bellow

Any other information on results incl. tables

Table: Radioisotope incorporation and ear weight:


        Radioisotope incorporation

Ear weight


Mean DPM


Mean (mg)





















Figures with asterisk (*) = values statistically significant on probability level < 0.05 (Mann-Whitney test)

Figures with cross(+) = values ≥ 3

NC – Negative control group

PC – Positive control group

DPM - Disintegrations Per Minute

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information
The positive control substance DNCB produced a positive LLNA response at an exposure level expected to give an increase in the Stimulation Index SI of ≥ 3 over the negative control group, which was in congruence with the expected mode of action of a contact allergen. The positive control also elicited a reaction pattern with a statistically significant increase in ear weight. The negative control did not reveal any changes. These results demonstrate that the method had sufficient sensitivity.

The animals exposed to the test substance at all dose levels showed no pathological skin reactions.
No symptoms of systemic toxicity were observed at all dose levels.
At the highest and middle dose levels, statistically significant increases in the weight of ears were recorded. The result of skin irritation effect was considered as positive – it means the test substance caused irritation of skin.

The comparison of the Stimulation Indexes between the treated groups and the control group revealed that the test substance caused a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes.

Executive summary:

The substance was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The Local Lymph Node Assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, Skin sensitization: Local Lymph Node Assay, Council Regulation (EC) No. 440/2008, published in O.J. L142, 2008.

In this study the contact allergenic potential of Hysperse 12 was evaluated after topical application to female BALB/c mice. Mice were exposed to three concentrations (50%, 5%, 0.5% (w/v)) of test substance suspended in vehicle (DAE 433) for 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated using radioactive labelling of proliferating cells. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.


positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and

test substance: 50%, 5%, 0.5% (v/v) in the solvent mixture, DAE 433.


Comparison of Stimulation Indexes between all treated groups and control vehicle group revealed that the test substance Hysperse 12 did not cause a significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. The Stimulation Index of

all treated groups is < 3.

The test substance Hysperse 12 did not cause increase of ear weight – it means the test substance did not cause irritation of skin.

The animals exposed to the test substance at all concentrations showed no pathological and no other negative clinical symptoms of intoxication throughout the experiment.

The positive control substance DNCB (concentration 0.5% (w/v) elicited a reaction pattern with statistically significant increase in Stimulation Index of cell proliferation and of ear weight, which was in congruence with his expected mode of action as a contact allergen.

Appropriate performance of the assay in the test laboratory was then demonstrated. In conclusion, at the given experimental conditions the test substance Hysperse 12 provided a negative result in LLNA study.