[N,N,N',N',N'',N''-hexaethyl-29H,31H-phthalocyaninetrimethylaminato(2-)-N29,N30,N31,N32]copper tris(dodecylbenzenesulphonate)

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:

December 2012 to March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study with well-characeterized test material

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Animal strain: Mouse / CBA/CaOlaHsd
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 - 12 weeks
- Mean weight at study initiation: ca. 18 - 23 g
- Housing: Single housing; Makrolon cage, type II
- Diet: STANRAB (P) SQC; SDS Special Diets Services, Altrip, Germany
- Water: Tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30- 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From16 January 2013 to 21 Jan 2013

Vehicle:

propylene glycol

Concentration:

25%, 10% and 5% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration, which could be technically used, was 25% (w/w). The dilutions were formulated in propylene glycol.

To determine the highest non-irritant test concentration, a pre-test (experimental conduct
in accordance with GLP but without a GLP status) was performed. Two mice per
concentration were treated epicutaneously with test item concentrations of 10 and 25 %
(w/w) each on three consecutive days. In the pre-test clinical signs were recorded after
each application as well as on day 5. Signs of local irritation were recorded on day 1, 2
and 5. Furthermore, the ears were punched after sacrifice at the apical area using a
biopsy punch (Ø 0.8 cm) and were immediately pooled per animal and weighed using an
analytical balance. Additionally the weight of the pooled lymph nodes from both sides was
determined for each animal.
Signs of systemic toxicity were not observed in the pre-test.
At the tested concentrations of 10 and 25% the animals showed no signs of local irritation
as confirmed by the ear weight measurements or ear thickness measurement.
Test item residues (blue colored ears) were observed in both test item groups.
After consulting the sponsor, the following dose levels were selected for the main study:
25%, 10% and 5% (w/w) in propylene glycol.

MAIN STUDY
TREATMENT SCHEME
Control group 1 Vehicle control in MEK
Test group 2 Test item 1% in MEK
Test group 3 Test item 2% in MEK
Test group 4 Test item 5% in MEK
Test group 5 Positive control 25% HCA in MEK

TREATMENT PREPARATION:
- Daily test item preparation and homogenization until end of each application period: The test item preparation will be produced for each application group shortly before application by stirring with a high speed homogenizer (Ultra-Turrax) and a magnetic stirrer.
- Test item homogenization until end of each application period: The homogeneity of the test item during application will be ensured by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the test item was suspended in the vehicle.

VEHICLE
- Vehicle: MEK (methyl ethyl ketone)
- Reason for the selection of the vehicle: MEK was used as the vehicle because good homogeneity of the preparation was achieved.
- Form of application: Suspension

EXPERIMENTAL PROCEDURE
- Route of application: Epicutaneously to the dorsum of both ears
- Application volume: 25 μL per ear
- Site of application: Dorsal surface of both ears
- Frequency of application: 3 consecutive applications (day 0 – day 2) to the same application site
- BrdU injection: On study day 4 mice were injected inter-peritoneally (i.p.) with 0.5 mL of 10 mg/mL BrdU in sterile phosphate-buffered physiological saline.
- Determination of ear weight: Immediately after sacrifice a circular piece of tissue (diameter 0.8 cm) was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal using an analytical balance. These measurements serve for detecting a potential inflammatory ear swelling.
- Removal and weight determination of the lymph nodes: Subsequently, the auricular lymph nodes (right and left) were dissected and total weight of both lymph nodes of each animal was determined.
- Preparation of cell suspension and determination of cell count: All lymph nodes (per test group) were carefully passed through a cell strainer and immediately afterwards, the cell suspension was diluted further with an isotone buffer solution and measured with a cell counter straight away.
- Measurement of BrdU in the lymph node cells: BrdU is measured by Elisa using a commercial kit (Roche Applied Science, Mannheim, Germany)

- Evaluation of results
The cell proliferation induced in the draining lymph node (auricular lymph node) after epicutaneous administration was used for evaluating the skin sensitizing potential. BrdU incorporation into the lymph node cells and the cell count, which was determined after cell isolation from both ear lymph nodes, indicates proliferation. Since certain skin irritants also induce cell proliferation and swelling of the draining lymph nodes of the application site, leading to false positive findings, ear weight as well as the lymph node weight was additionally determined. Especially the ear weight, which is a surrogate for ear swelling, was used as an indicator of irritation.
The effects of test item treatment on BrdU incorporation, cell count of lymph node cells, weight of the lymph nodes as well as ear weight in the test groups was assessed in comparison to the vehicle control group.

CLINICAL EXAMINATIONS
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- Signs and symptoms: Obvious signs of systemic toxicity and/or local inflammation at the application sites was checked individually.
- Mortality: A check for moribund and dead animals was made at least once each workday.

CLINICAL PATHOLOGY
- No examinations were performed.

PATHOLOGY
- The animals were sacrificed on study day 5 about 24 hours after BrdU injection by cervical dislocation. Necropsies were not be performed.
- Animals, which died spontaneously during the observation period, were necropsied as soon as they are found dead and any abnormalities was recorded.

CALCULATION OF RESULTS
- Results for each treatment group are expressed as the mean SI. The SI is derived by dividing the mean BrdU labelling index/mouse within each test substance group and the PC group by the mean BrdU labelling index for the NC group. The average SI for the NCs is then one.
The BrdU labelling index is defined as: BrdU labelling index = (ABSem – ABS blankem) – (ABSref – ABS blankref)
Where; em = emission wavelength; and ref = reference wavelength.
- A result is regareded as positive when SI ≥ 1.6.

Positive control substance(s):

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

Statistics:

Calculation of mean and standard deviation was performed.

Positive control results:

See table 1.

Parameter:

SI

Remarks on result:

other: SI = 1.0 (25%), 1.1 (25%) and 1.1 (5%)

Table 1 Stimulation Index. BrdU labeling Index

a) Mean BrdU labeling index was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals per group)

^b SI =Stimulation Index (values of the test item groups related to the mean value of the control group)

ABS    =  absorbance

*    statistically significant increase vs. control group (p<0.05)

Test Group	Animal No.	ABS450- ABS blank450	ABS690- ABS blank690	BrdU labeling Index	Mean BrdU labeling Index	S.I.^b)	Group S.I.
1  Vehicle Control	1	0,045	0,002	0,043	0,076	0,6	1,0
	2	0,079	0,001	0,078		1,0
	3	0,094	0,000	0,094		1,2
	4	0,111	0,002	0,110		1,5
	5	0,055	0,002	0,053		0,7
	6	0,090	0,001	0,089	0,082	1,2	1,1
2	7	0,086	0,005	0,081		1,1
5%	8	0,041	0,002	0,039		0,5
	9	0,096	0,004	0,092		1,2
	10	0,112	0,005	0,107		1,4
	11	0,106	0,004	0,103	0,084	1,4	1,1
3	12	0,097	0,005	0,093		1,2
10%	13	0,096	0,005	0,091		1,2
	14	0,097	0,004	0,094		1,2
	15	0,047	0,005	0,042		0,6
	16	0,082	0,005	0,078	0,076	1,0	1,0
4	17	0,108	0,005	0,103		1,4
25%	18	0,070	0,004	0,066		0,9
	19	0,064	0,006	0,058		0,8
	20	0,080	0,005	0,075		1,0
25 %  HCA in propylene glycol	21	0,343	0,004	0,340	0,337*	4,5	4,5
	22	0,270	0,006	0,265		3,5
	23	0,486	0,004	0,483		6,4
	24	0,324	0,003	0,321		4,3
	25	0,283	0,006	0,277		3,7

 

Table 2 Ear thickness

Animal No:	Concentration	Ear Thickness
		prior to 1^stApplication			prior to 3^rdApplication			prior to Necropsy			Difference	Ear Swelling	Difference	Ear Swelling
		(mm)			(mm)			(mm)			Day 1 to    Day 2	Day 2	Day 1 to            Day 5	Day 5
		Right Ear	Left	Mean	Right Ear	Left Ear	Mean	Right Ear	Left Ear	Mean
			Ear								(mm)	(%)	(mm)	(%)
1	25% (w/w)	0,21	0,22	0,22	0,23	0,23	0,23	0,24	0,25	0,25	0,02	6,98	0,03	14,0
2		0,24	0,23	0,24	0,25	0,23	0,24	0,25	0,25	0,25	0,01	2,13	0,02	6,4
3	10% (w/w)	0,24	0,23	0,24	0,23	0,23	0,23	0,25	0,25	0,25	-0,005	-2,13	0,015	6,4
4		0,23	0,23	0,23	0,23	0,23	0,23	0,24	0,23	0,24	0	0,00	0,00	2,2
5	propylene glycol	0,23	0,23	0,23	0,23	0,23	0,23	0,23	0,23	0,23	0	0,00	0,00	0,00
6		0,23	0,23	0,23	0,23	0,22	0,23	0,23	0,22	0,23	-0,005	-2,17	-0,01	-2,17

Table 3: Body weights

Animal No.	Concentration	Body Weight
		prior	prior	Difference	Difference
		1^stApplication	to Sacrifice (Day 5)	Day 0 to Day 5	%
		(g)	(g)	(g)
1	25%	21,5	21,0	-0,5	-2,3
2		21,8	20,9	-0,9	-4,1
3	10%	22,1	21,6	-0,5	-2,3
4		19,8	20,2	0,4	2,0
5	Propylene glycol	19,3	19,4	0,1	0,5
6		20,1	20,9	0,8	4,0

 

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A local lymph node assay was performed using test item concentrations of 5, 10 and 25% (w/w; weight per weight) in propylene glycol (Bioassay 2013). Doses based on previously performed one pretest in the same animal strain in which 6 mice in total were used.

In the pre-test the highest concentration that could be technically used was 25% (w/w). Signs of systemic toxicity were not observed in the pre-test. At the tested concentrations of 10 and 25% the animals showed no signs of local irritation as confirmed by the ear weight measurements or ear thickness measurement. Test item residues (blue colored ears) were observed in both test item groups.

In the main study the animals did not show any relevant signs of systemic toxicity during the course of the study and no cases of mortality were observed. Both in the low, mean and high dose group test item residues (blue coloured ears) were observed on day 2, 3 and 5. A statistically significant increase in ear weights was observed in the high dose group (25%) in comparison to the vehicle control group, without reaching biological relevance.Furthermore, the cut-off-value for a positive response regarding the ear weight index of 1.25 was not exceeded in any dose group. In this study Stimulation Indices (S.I.) of 1.1, 1.1 and 1.0 were determined with the test item at concentrations of 5, 10 and 25 % (w/w) in propylene glycol.

As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight, lymph node cell count and ear weight measurement was determined in the positive control.

Migrated from Short description of key information:
The colourant did not cause skin sensitization in the LLNA in mice (BASF 2013). The study followed OECD testing guideline 442B of July 22, 2010 and GLP:

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for skin sensitization under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).