Bis and tris and tetra (4-{bis[4-(dimethylamino)phenyl]methylene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium) [12,21-dihydro-29H,31H-phthalocyanine-bis and tris and tetrasulfonato-k4N29,N30,N31,N32]cuprate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The reliability is rated 2 because the study followed the standard guideline of reference (OECD 429), which describes a procedure designed to evaluate this endpoint, but with an important protocol deviation. The results were reviewed for reliability and assessed as valid, and the study was conducted under GLP condition.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

: Use of the lymph node cell count for measurement of the cell proliferation instead of the determination of DNA synthetis (3H-thymidine incorporation into the lymph node cells).

Principles of method if other than guideline:

Lymph node cell count is more direct measurement of cell proliferation than determination of DNA synthesis and therefore considered an appropriate parameter for evaluation of cell proliferation in the assay. In this case, the cut-off value is much lower and it's fixed at 1.4 times increase of stimulation index (instead of 3 for the 3H-thymidine method). This is understandable by the facts that cell count indices have:
- Lower individual variance compared to 3H-thymidine incorporation
- Lower maximum stimulation indices compared to radioactive labelling.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 8 to 10 weeks old
- Weight at study initiation: 19.7 to 23.2 g
- Housing: Individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet : Food (M20, SDS) ad libitum
- Water : Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 15 changes /hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Vehicle:

dimethylformamide

Concentration:

Test item diluted at concentrations of 50% (v/v), 25% (v/v) and 10% (v/v) in the dimethylformamide (DMF).

No. of animals per dose:

4 animals per dose + 4 animals for the vehicle control (vehicle alone) : Pooled lymph nodes approach.
(An additional mouse was treated in each group in case of problem which may occur during the study, in particular during the excision of lymph nodes. As no problem occurred during the test, the lymph nodes of the additional mouse were not collected and only the data concerning the 4 first animals of each group were used in the study).

Details on study design:

RANGE FINDING TESTS
- Concentration: 25 µL of the test item diluted at 50% in DMF to the dorsal surface of each ear for three consecutive days (D1, D2 and D3).
- Irritation: Daily examination. Ear thickness recorded on D1, D3 and D6.
- Lymph node proliferation response: Not performed.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Pooled approach with the lymph node cell count method.
- Criteria used to consider a positive response: In order to assess any posible irritant effect 1) Ear thickness measurements of the right ear of each animal (vehicle and treated groups), by a micrometer were performed on D1, D3 and D6; 2) Any local reactions (irritation reaction or any other observation) were recorded. 3) On D6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighed and the two lymph nodes per mouse were weighed. In fact, possible irritancy may be involved in false positive lymphoproliferative responses.

In order to assess the skin sensisation of the test item, animals were anaesthetised on D6. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. The proliferation response of lymph node cells was expressed as the stimulation index :

SI = Cell count of treated group / Cell count of control group

The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values. The irritation level were also taken into account for the interpretation of the results. Any test item failing to produce a SI<1.4 will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the appropriate concentration of the test item (10%, 25% , 50% or the vehicule) was applied to the dorsal surface of each ear, using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. The application was repeated on days 2 and 3. On D6, the draining auricular lymph nodes from mouses were excised and pooled for each experimental group after anaesthetisia. Then, a single cell suspension of the lymph node cells of 4 mice of each group was prepared by gentle mechanical tissue disaggregation through a 200-mesh cell strainers in 4 mL of PBS containing 0.5% BSA into a 6-well multiwell plate. 10 µL of this cell suspension was diluted in 10 mL of physiological saline solution (NaCl 0.9%). The lymphocyte cells were counted using a cell counter. For the run, the lower size selected was 5 µM and the upper size selected was 15 µm (the average size of a lymphocyte is 8 µm).

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Positive control results:

CURRENT POSITIVE CONTROL STUDY

Group 1: AOO – 21.11 x 106 cells/mL – SI: n.a.
Group 2: 5% - 25.61 x 106 cells/mL – SI = 1.21  Negative
Group 3: 10% - 31.12 x 106 cells/mL – SI = 1.47  Positive
Group 4: 25% - 50.87 x 106 cells/mL – SI = 2.41  Positive


The EC1.4 value = 8.65%

In conclusion, in view of these results, under these experimental conditions, the positive control must be classified R43 "may cause sensitisation by skin contact" in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45. This substance must be characterised by the symbol "Xi" and the warning label "Irritant". In accordance with the regulation (EC) n°1272/2008, the test item must be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

Parameter:

SI

Remarks on result:

other: SI = Cell count of treated group / Cell count of control group. Calculated by the pooled approach Group 2, SI = 1.66 Group 3, SI = 1.79 Group 4, SI = 3.01

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The disintegrations per minute wasn't performed. A cell counting deviation was used: Group 1 (DMF) = 17.37 x E6 cells/mL Group 2 (10%) = 28.78 x E6 cells/mL Group 3 (25%) = 31.14 x E6 cells/mL Group 4 (50%) = 52.20 x E6 cells/mL

Cell count, stimulation index and calculation of EC1.4

Groups	Test item	Cell count/groups (x10^6cells/mL)	Stimulation Index (S.I.)	Result	EC1.4value
1	DMF	17.37	n.a.	n.a.	n.a.
2	10%	28.78	1.66	Positive	n.a.
3	25%	31.14	1.79	Positive
4	50%	52.20	3.01	Positive

 

The EC_1.4 value is determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.4-fold threshold, according the equation:

EC_1.4= c + [(1.4 – d) / (b – d)] x (a – c)

a= the lowest concentration giving stimulation index > 1.4

b= the actual stimulation index caused by a.

c= the highest concentration failing to produce a stimulation index of 1.4

d= the actual stimulation index caused by c.

Since no concentration failing to produce a stimulation index of 1.4 was find, the EC_1.4value can not be determined in this study.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In view of these results, under these experimental conditions, the test item Sepisol Fast Violet 3B must be classified as a skin sensitiser. In, accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45, this item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact". In accordance with the regulation with the Regulation EC N° 1272/2008, the test item must be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

Executive summary:

The test was performed to assess the skin sensitisation potential of the test item Sepisol Fast Violet 3B in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

A preliminary study was performed using one mouse, treated daily (on D1, D2 and D3) with 25 µL per ear of the test item diluted at 50% in dimethylformamide (DMF). After a daily examination until D6, any signs of toxicity or excessive local irritation observed during this period were recorded. Therefore, the concentration of 50% was chosen as the highest concentration for the main study.

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

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cell counting. The experimental protocol was established according the OECD guideline N° 429 dated 22 july 2010 and the test method B.42 of the council regulation N° 440/2008.

No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. A blue coloration of the treatment site was noted from day 2 in all treated animals.

No significant increase in ear thickness and in ear weight was noted in none of animals.

Therefore, the test item must be considered as not excessively irritant at the three concentrations.

The stimulation indes (SI) calculated by pooled approach was 1.66, 1.79 and 3.01 for the treated groups at 10%, 25% and 50% respectively.

The EC_1.4value can not be determined in this study.

In view of these results, under these experimental conditions, the test item Sepisol Fast Violet 3 must be classified as a skin sensitiser. In accordance with the criteria for classification, packaging and labelling of dangerous substances and preparation of the E.E.C. Directives 67/548, 2001/59 and 99/45, this item must be characterised by the symbol "Xi" and the warning label "Irritant" with the risk sentence R43 "May cause sensitisation by skin contact".

In accordance with the Regulation EC N°1272/2008, the test item must be classified in category 1 "Skin sensitisation". The signal word "Warning" and hazard statement H317 "May cause an allergic skin reaction" are required.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The sensitization test was performed according to the OECD guideline 429, using the cell counting method to determined the proliferation of lymphocytes in the draining auricular lymph nodes instead of the radioactive method [³H]-methyl thymidine.

The positive control used to assess the sensitivity of the strain of mouse used was the α-Hexylcinnamaldehyde.

Inter and Intra-laboratory validation of EC values was performed on this positive control to assess the performance of the test based on the cell counting method (see graphs 1 and 2 in the attached documents part).

Three groups were dosed with 10, 25 and 50% of Sepisol Fast Violet 3B, but since the minimal dose (10%) gives a calculated stimulation index > 1.4, no calculated EC_1.4 by linear interpolation of points on the dose-response curve value could be determined.

Based on the available data, a theoretical EC_1.4 value has been derived in order to better define the classification (category 1A or 1B).

At a concentration of 0%, the cell count/group is considered to be at least equal to the one of the negative control, which leads to a stimulation index of 1. Stimulation index curve of the Sepisol Fast Violet 3B and of the positive control are presented in the graph 3. The linear trendlines at 0% are effectively intersecting the zero ordinate axis towards the value of 1 in both cases.

By taking the average of the 2 points 1 and 1.66, the theoretical EC_1.4 value is equal to [10/(1.66 -1)] x (1.4 -1)= 6%. This theoretical value of 6% is confirmed graphically (graph 4). By including the mean value to draw the curve on the graph 4, i.e. an stimulation index of 1.33 (mean of 1 and 1.66 stimulation indexes) for a concentration of 5% (mean of 0 and 10%), the 1.4 cut-off line cuts the Sepisol Fast Violet 3B curve at 6%.

As the 6% value is not close of the 2% cut off value for classifying the substances into the category 1A "skin sensitisation", the Sepisol Fast Violet 3B is considered category 1B "skin sensitisation", and the value of 6% was chosen to perform the risk characterisation assessment.

Migrated from Short description of key information:
Justification of the classification category 1B "skin sensitisation"

Justification for selection of skin sensitisation endpoint:
The study used to evaluate this endpoint followed the standard guideline reference (OECD 429). Despite the protocole deviation, the key study is considered reliable to assess this endpoint.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The theoretical EC_1.4value is equal to 6% which is above the 2% cut off value for classifying the substances into the category 1A "skin sensitisation". Therefore, the Sepisol Fast Violet 3B is classified category 1B "skin sensitisation".