A reaction mixture containing the following components;A. Tetralithium [2(or 3), 9 (or 10), 16 (or 17), 23 (or 24)-tetrakis (3-sulfonato-propylsulfonyl) phthalocyaninato]cupurate (II)B. Trilithium [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl]tris (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)C.Dilithium bis [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] bis (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)D. Lithium tris [3-(2-hydroxylpropylsulfamoyl)propylsulfonyl] (3-sulfonato-propylsulfonyl)phthalocyaninatocupurate (II)E. Tetrakis [3-(2-hydroxpropylsulfamoyl) proppylsulfonyl]phthalocyaninatocupurate (II)In the ratio A:B:C:D:E 6.25 : 25 : 37.5 : 25 : 6.25

Skin sensitisation

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Administrative data

Endpoint:

skin sensitisation, other

Remarks:

in vivo, LLNA, existing study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was performed between 14 January 2003 and 20 January 2003. The final report was issued 11 February 2003.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Meets the criteria for classification as reliable without restriction according to Klimisch et al (1997)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Sixteen female CBA/Ca (CBA/CaBkl) strain mice were supplied by a reptuable supplier. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. At the start of the study the animals were in the weight range of 15 to 23 g, and were eight to twelve weeks old. After an acclimatisation period of at least five days, each animal was selected at random and given a number unique within the study which was written on the tail using a black indelible marker pen.The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes. Free access to mains tap water and food was allowed throughout the study.The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study

Study design: in vivo (LLNA)

Vehicle:

other: 0.5% Tween 80 in distilled water

Concentration:

0.1%, 1% or 10% w/w

No. of animals per dose:

four

Details on study design:

MethodsThree groups, each of four animals, were treated with 50 µl of the test material (25 µl per ear) as a solution in 0.5% Tween 80 in distilled water at concentrations of 0.1%, 1% and10% wlv. A further group of four animals was treated with 0.5% Tween 80 in distilled water alone. Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 0, 1, 2). The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner. Five days following the first topical application of the test material (Day 5) all mice were injected via the tail vein with 250 µl of phosphate buffered saline containing 3H-methyl thymidine (3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, Amersham Pharmacia Biotech UK Ltd) giving a total of 20 µCi to each mouse.ObservationsClinical Observations: All animals were observed on a daily basis. Any signs of toxicity or signs of ill health during the study were recorded.Bodyweights: The bodyweight of each mouse was recorded on Day 0 (prior to dosing) and Day 5 (prior to termination).Terminal ProceduresTermination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 ml of phosphate buffered saline (PBS) was added to the pooled lymph nodes.Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 ml of PBS into a petri dish labelled with the project number and dose concentration. The lymph node cells suspension was transferred to a 10 ml centrifuge tube. The petri dish was washed with an additional 5 ml of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 G) for ten minutes, washed with 10 ml of PBS, re-pelleted at 1400 rpm and precipitated with 3 ml of 5% trichloroacetic acid (TCA).Determination of 3HTdR Incorporation: After overnight incubation at 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for ten minutes, re-suspended in 1 ml of TCA and transferred to 10 ml of scintillation fluid (Optiphase 'Trisafe'). 3HTdR incorporation was measured by scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left for approximately twenty minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately twenty minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman 186500 scintillation system (Beckman Instruments Inc, Fullerton, CA).

Positive control substance(s):

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

Statistics:

none

Results and discussion

Positive control results:

Positive control studies conducted in the laboratory responded as expected and confirmed that the test system was operating as expected.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 0.78

Test group / Remarks:

0.1% w/w

Results 2

Key result

Parameter:

SI

Value:

ca. 0.52

Test group / Remarks:

1% w/w

Results 3

Key result

Parameter:

SI

Value:

ca. 0.63

Test group / Remarks:

10% w/w

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test material was considered to be a non-senstiser under the conditions of the test.

Executive summary:

Introduction

A study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of OECD Guideline for the Testing of Chemicals, No 429 2002 "Skin Sensitisation: Local Lymph Node Assay"

Results

The simulation index was recorded for three concentrations of the test material at concentrations of 0.1,1 and 10 % w/w and the SI recorded at each concentration was 0.78, 0.52 and 0.63 respectively.

Conclusion

The test material was considered to be a non-sensitiser under the conditions of the test.