Copper chlorophthalocyanine

Endpoint summary

Description of key information

The substance is considered to be non sensistizing based on read-across in GLP and OECD guideline 429 compliant studies with source substances.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Specific details on test material used for the study:

- Substance type: blue solid
- Analytical purity: no data given
- Lot/batch No.: KRON 200106
- Purity> 85%
- Expiration date of the lot/batch: 31-Mar-2006
- Stability under test conditions: stable under storage conditions
- Storage condition of test material: in the original container at room temperature (20 °C +- 3 °C), away from direct sunlight

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g (ordered)
- Housing: individually in Makrolon type-2 cages with standard softwood bedding
- Diet: Pelleted Standard Kliba 3433, batch no. 78/03 mouse maintainance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: under test conditions after haelth examination; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 30 - 70 %
- Air changes per hr: 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

dimethyl sulphoxide

Concentration:

2.5, 5 and 10 %

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP test was conducted in 2 mice with concentrations of 1, 2.5, 5 and 10 %. In the main study 3 consecutive concentrations were assayed. The top dose was the highest achievable concentration whilst avoiding systemic toxicity and excessive local irritation. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY:
Each test group was treated by topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations. The application volume, 25 µl, was spread over the entire dorsal surface (ca. 8 mm diameter) of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear´s surface to prevent the loss of any of the test item applied.
Five days after the first topical application, all mice were administered with 250 µl of 79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tailvein.
Approx. 5 h after treatment with 3HTdR all mice were euthanized with dry ice (CO2). The draining lymph nodes were rapidly excised and pooled for each experimental group (8nodes/group). Single cell suspensions of pooled lymph node cells were prepared. Cells were resuspended in 5 % trichloroacetic acid ans incubated at 4 °C for at least 18 h to precipitate the macromolecules. The precipitates were resuspended in 5 % trichloroacetic acid and transferred to glass scintillation vialscontaining 10 ml scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was thenmeasured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test group relative to the recorded control group (Stimulation Index S.I.). Before values were determined, mean scintillation background DPM was subtracted from data obtained.
In addition, mortality/viability (twice daily), body weights (priot to first application and prior to necropsy) and clinical signs (local/systemic, daily with particular attention to the treatment sites) were determined.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle was quantitatively added. The w/v dilutionswere prepared individually using a magnetic stirrer as homogenizer.
Test item solutions were made freshly before each dosing occasion and no more than 4 h prior to application.
Homogeneity was maintainedduring treatment with the magnetic stirrer.

Positive control substance(s):

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

Statistics:

A test item was considered as sensitizer if the following criteria were fulfilled:
- Exposure to at least one concentration resulted in an incorporation of 3HTdR of at least 3-fold or greater than in controls as indicated by S.I.
- Data were compatible with a conventional dose-response, although allowance must be for either local toxicity or immunological suppression.

For statistical analysis, the mean values and standard deviations were calculated in the body weight tables.

Parameter:

SI

Value:

0.8

Test group / Remarks:

2.5%

Parameter:

SI

Value:

1.1

Test group / Remarks:

5%

Parameter:

SI

Value:

1

Test group / Remarks:

10%

Cellular proliferation data / Observations:

The background DPM was measured twice and was 3 or 10, respectively. The dpm, determined for the control group, was 4593, for the 2.5 % group it was 3672, for the 5.0 % group it was 5122 and for the 10.0 % group it was 4488.

No deaths occured during the study period.

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

The body weight of the aninals was within the normal range recorded for animals of this strain and age. One animal of the 10 % test item concentration group lost weight during the study, but this was considered to be incidental.

Test item concentration % (w/v)		dpm measurement	dpm - background	number of lymph nodes	dpm per lymph node	S.I.
	background	10
	background	3
	control group	4593	4586	8	573
2.5	test group	3679	3672	8	459	0.8
5.0	test group	5129	5122	8	640	1.1
10.0	test group	4495	4488	8	561	1.0

The positive control, alpha-hexylcinnamaldehyde, was tested in concentrations of 5.0, 10.0 and 25.0 % and produced S.I.s of 1.5, 2.3 and 8.4, respectively.

Interpretation of results:

GHS criteria not met

Conclusions:

In this study stimulation indices of 0.8, 1.1 and 1.0 were determined with the test material at concentrations of 2.5, 5 and 10 % (w/v), respectively in DMSO.
Under the experimental conditions chosen, the test material was not found to be a sensitizer when tested at up to the highest achievable concentration of 10 % (w/v) in DMSO.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Name of test material (as cited in study report): CT-BX 121
- Substance type: blue powder
- Analytical purity: >99%
- Lot/batch No.: SF20927
- Storage condition of test material: room temperature in the dark

Species:

mouse

Strain:

other: CBA/CaBkl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: B & K Universal Ltd, Hull, UK
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: individually in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet: Certified Rat and Mouse Diet, Code 5LF2 (BCM IPS Ltd., London, UK), ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 - 25 °C
- Humidity: 30 - 70 %
- Air changes: 15 changes per hr
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

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

No. of animals per dose:

four animals per dose

Details on study design:

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 3 consecutive days. The test material was administered by using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of 4 animals received the vehicle anlone in the same manner.
Five days following the first topical application of the test material, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR, 80 µCi/ml, specific activity 2.0 µCi/mmol; Amersham Biosciences UK Ltd.), giving a total of 20 µCi to each mouse.
Animals were observed post dose on day 1, twice daily on days 2 and 3 and on a daily basis on days 4, 5 and 6. Any signs of toxicity or ill health were recorded. The body weight of the animals was determined on day, 1 prior to dosing, and on day 6, prior to termination.
5 hours following the administration of 3HTdR all animals were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the mice were excised and pooled for each animal group. For each group, 1 ml of PBS was added to the pooled lymph nodes.
A single cell suspension of the pooled lymph node cells was prepared by gentle mechanic disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze and collected into a centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (ca. 190 g) for 10 min. The pellet was resuspended in PBS and re-pelleted. To precipitate out the radioactive material, the pellet was resuspenden in 3 ml of 5 % Trichloroacetic acid (TCA).
After overnight incubation at 4 °C the precipitates were removed by centrifugation at 2100 rpm (ca. 450 g) for 10 min, resuspended in 1 ml TCA and transferred to 10 ml scintillation fluid (Optiphase, "Trisafe"). 3HTdR incorporation was measured by ß-scintillation counting. The number of radioactive disintegrations per minute was then measured using a Beckman LS6500 scintillation system.

The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). The test material will be regarded as sensitizer, if at least one of the tested concentrations results in threefold greater increase in 3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as non-sensitizer.

Positive control substance(s):

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

Parameter:

SI

Value:

1.32

Test group / Remarks:

5%

Parameter:

SI

Value:

1.65

Test group / Remarks:

10%

Parameter:

SI

Value:

1.49

Test group / Remarks:

25%

Cellular proliferation data / Observations:

DPM data:
Test group 1: vehicle (acetone/olive oil 4:1): 683.38 dpm/node
Test group 2: 5 % test substance in vehicle: 899.50 dpm/node
Test group 3: 10 % test substance in vehicle: 1131.00 dpm/node
Test group 4: 25 % test substance in vehicle: 1020.05 dpm/node

There were no deaths. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study.

The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period.

A stimulation index of less than 3 was recorded for the three concentrations of the test material.

The positive control alpha-hexylcinnamaldehyde provided positive results and was considered to be a sensitizer under the experimental conditions of the test.

Interpretation of results:

GHS criteria not met

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Specific details on test material used for the study:

Green solid
Batch DEBF 015527
Stable under storage conditions
Expiry data 31-MAR-2006

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 16 g - 24 g (ordered)
- Housing: individually in Makrolon type-2 cages with standard softwood bedding
- Diet: Pelleted Standard Kliba 3433, batch no. 78/03 mouse maintainance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: under test conditions after haelth examination; only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 30 - 70 %
- Air changes per hr: 10 - 15
- Photoperiod: 12 hrs dark / 12 hrs light

Vehicle:

dimethyl sulphoxide

Concentration:

5, 10 and 25%

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP test was conducted in 2 mice with concentrations of 5, 10 and 25 %. The top dose is the highest technically achievable concentration.

MAIN STUDY:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5 %, 10 % and 25 % (w/v) in dimethylsulfoxide (DMSO). The application volume, 25 µI, was spread over the entire dorsal sutiace (0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent the loss of any of the test item applied. Five days after the first topical application, all mice were administered with 250 µI of
79.6 µCi/ml 3HTdR (equal to 19.9 µCi 3HTdR) by intravenous injection via a tailv ein.
Approximately five hours after treatment with 3HTdR all mice were euthanized with dry ice (C02).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3 HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Positive control substance(s):

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

Statistics:

A test item was considered as sensitizer if the following criteria were fulfilled:
- Exposure to at least one concentration resulted in an incorporation of 3HTdR of at least 3-fold or greater than in controls as indicated by S.I.
- Data were compatible with a conventional dose-response, although allowance must be for either local toxicity or immunological suppression.

For statistical analysis, the mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Results with the latest postive control study are attached to the report (RCC Study Number 852080),

Parameter:

SI

Value:

1.4

Test group / Remarks:

5%

Parameter:

SI

Value:

1.3

Test group / Remarks:

10%

Parameter:

SI

Value:

1.3

Test group / Remarks:

25%

Cellular proliferation data / Observations:

No deaths occured during the study period.
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.
The body weight of the aninals was within the normal range recorded for animals of this strain and age.

Test item concentration %( w/v)		Measurement dpm	Calculation			Result
			dpm - BG a)	number of lymph nodes	dpm per lymph node b)	S.I.
--	BG1	10	--	--	--	--
--	BG II	3	--	--	--	--
--	CG 1	5238	5231	8	654	--
5	TG 2	7418	7411	8	926	1.4
10	TG 3	6791	6784	8	848	1.3
25	TG 4	6816	6809	8	851	1.3

BG=Background (1 ml 5 % trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a)      = The mean value was taken from the figures BG I and BG II

b)      = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The positive control, alpha-hexylcinnamaldehyde, was tested in concentrations of 5.0, 10.0 and 25.0 % and produced S.I.s of 1.5, 2.3 and 8.4, respectively.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The following summarizes the studies performed with the source chemicals. The read-across justification is attached to the endpoint study record.

CAS No. 147-14-8 (two OECD 406, one OECD 429):

An in vivo study with 10 female guinea pigs in the test group and 5 female guinea pigs as control group was conducted, according to the method described by Magnusson and Kligman. The intradermal induction (6 injections in groups of two per animal) was conducted with FCA, the test material 7.5 % (w/v) in Alembicol D and the test material 7.5 % (w/v) in a mixture of FCA and Alembicol D. Epicutaneous induction (one week after intradermal induction) was conducted with 50 % of the test material in Alembicol D. The challenge (14 days after epicutaneous induction) was performed with 25 % and with 50 % of the test material in Alembicol D (acc. OECD 406, GLP; Huntingdon 2001). No animal in the control group and no animal in the treated group exhibited positive reactions 24 or 48 hours after the challenge procedure.

Skin sensitizazion testing was performed in female guinea pigs according to the method of MAGNUSSON & KLIGMAN (OECD 406) and under GLP.Intradermal induction was performed using 5 % Pigment Blue 15 in sesam oil (Aventis 2002). Dermal induction and challenge treatment were carried out with 25 % Pigment Blue 15 in sesam oil.The validity of the test system is confirmed by the periodically conducted positive control test using alpha-hexylcinnamaldehyde for the maximization test.Based on the results of this study Pigment Blue 15 showed no evidence for sensitizing properties according to the classification criteria of Directive  2001/59/EC.

In another study, a local lymph node assay was conducted according to OECD 429 and under GLP conditions (Safepharm 2004). Four female CBA/CaBkl mice per dose (5 %, 10 % or 25 % w/w of the test material in acetone/olive oil 4:1 v/v) were treated by daily application of 25 µl of the appropriate concentration of either the test material or with vehicle alone to the dorsal surface of each ear for 3 consecutive days. Five days following the first topical application, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 20 µCi 3H-methyl thymidine (3HTdR) to each mouse. Animals were observed post dose, any signs of toxicity or ill health were recorded. The body weight of the animals was determined prior to dosing and prior to termination. 5 hours following administration of 3HTdR all animals were killed. The draining auricular lymph nodes were excised, pooled for each group and a single cell suspension of the cells was prepared. 3HTdR incorporation was measured by ß-scintillation counting. The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). There were no early deaths during the study. No signs of systemic toxicity was noted in the test or in the control animals during the study. Blue-coloured staining on the ears, face, feet and fur was noted in all test animals during the study. The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period. A stimulation index of less than 3 was recorded for the three concentrations of the test material, indicating no sensitizing potential of the tested material.

 

CAS No. 1328-53-6:

A local lymph node assay was conducted according to OECD 429 and under GLP conditions (RCC 2004). Three groups each of four female mice were treated daily with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in dimethylsulfoxide (DMSO) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically achievable concentrationinthe vehicle. A control group of four mice was treated with the vehicle (dimethylsulfoxide (DMSO)) only. Five days after the first topicalapplicationthe mice were injected intravenouslyintoa tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymp node cells was determined by the incorporation of 3H-methyl thymidine measured in aß-scintillation counter.

No clinical signs were observed.All treated animals survived the scheduled study period. The results obtained (STIMULATION INDEX (S.I.)) were 1.4, 1.3 and 1.3 for all three test groups.

CAS No. 27614 -71 -7:

A local lymph node assay was conducted according to OECD 429 and under GLP conditions (RCC 2004). Groups of four female CBA mice per dose (5 %, 10 % or 25 % w/w of the test material in DMSO) were treated by daily application of 25 µl of the appropriate concentration of either the test material or with vehicle alone to the dorsal surface of each ear for 3 consecutive days. Five days following the first topical application, all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing 19.9 µCi 3H-methyl thymidine (3HTdR) to each mouse. Animals were observed post dose, any signs of toxicity or ill health were recorded. The body weight of the animals was determined prior to dosing and prior to termination. 5 hours following administration of 3HTdR all animals were killed. The draining auricular lymph nodes were excised, pooled for each group and a single cell suspension of the cells was prepared. 3HTdR incorporation was measured by ß-scintillation counting. The proliferation response of the lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into the lymph node cells of test nodes relative to that recorded for the control nodes (stimulation index). There were no early deaths during the study. No signs of systemic toxicity was noted in the test or in the control animals during the study. The body weight changes of the test animals during the study were comparable to those observed in the corresponding control animals over the same time period (except for one animalof the 10 % test item concentration group which lost weight during the study, but this was considered to be incidental). No dose-response relation was observed and a stimulation index of less than 3 was recorded for the three concentrations of the test material. The test item concentration of 2.5 % produced a S.I. of 0.8, a concentration of 5.0 % produced a S.I. of 1.1 and a concentration of 10.0 % produced a S.I. of 1.0.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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. The stimulation indices in the LLNA (OECD 429) did not show a dose dependent increase. No EC3 could be established. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No 2015/1221.