Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed GLP and OECD guideline study

Cross-reference

Reason / purpose for cross-reference:

exposure-related information

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17.3 - 22.5 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Vehicle:

dimethylformamide

Concentration:

5, 10, and 25%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:

To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest (study 1093403) was performed in two mice (pretest excluded from Statement of Compliance). The data showed that the highest test item concentration, which could be technically used, was a 25 % solution in DMF. In other vehicles tested, e.g propylene glycol, actone:olive oil (4+1), methyl ethyl ketone, ethanol (70%), and DMSO, a higher concentration could not be achieved. At the 25 % dilution the treated mouse did not show any signs of systemic toxicity.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.



MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 5, 10, and 25% in DMF. The application volume, 25 µL was spread over the entire dorsal surface (~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL THYMIDINE AND DETERMINATION OF INCORPORATED 3H-METHYL THYMIDINE

Five days after the first topical application, all mice were intraveneously injected into a tail vein with radio-labelled thymidine (3HTdR). Approximately five hours after treatment with 3HTdR all mice were sacrificed and the draining lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed with phosphate buffered saline and incubated with trichloroacetíc acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a scintillation counter.

INTERPRETATION OF RAW DATA

The proliferation 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 lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node values were determined, mean scintillation-background DPM was subtracted from test and control raw data. A test item is regarded as a sensitiliser in the LLNA if the following criteria are fulfilled:
-First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
-Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

OBSERVATIONS

In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:

Mortality / Viability: At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables.

Positive control results:

Experiment performed in May 2007 using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 2.43, 4.07, 4.88, respectively.
The EC3 value calculated was 6.7 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see depicted table below

Calculation and results of individual data; Vehicle: DMF

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
24	---	---	---	---	24	---
24	---	---	---	---	24	---
2268	2244	8	280.5	1.00	2268	2244
7330	7306	8	913.3	3.26	7330	7306
7792	7768	8	971.0	3.46	7792	7768
5554	5530	8	691.3	2.46	5554	5530

1    =  Control Group

2-4=  Test Group

^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 EC3 value could not be calculated, since an inverse dose relationship was observed.

 

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

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

BODY WEIGHTS

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:

other: inconclusive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

The test item was found to be a skin sensitiser under the test conditions of this study. However, taking into account the inverse dose relationship and the borderline result obtained for the high dose of 25%, it cannot be excluded that the test item would show sensitising properties at higher concentrations or interferes with the test system in general. Thus, the result of this study remains inconclusive.

Executive summary:

In order to study a possible skin sensitising potential of the test item, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in DMF by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (DMF) only. Five days after the first topical application the mice were injected intravenously into a 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 lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 3.26, 3.46, and 2.46 were determined with the test item at concentrations of 5, 10, and 25% in DMF. A dose response was not observed. The EC3 value could not be calculated, since an inverse dose relationship was observed.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The OECD 406 method was employed because the OECD 429 was proven as "not feasible" due to the experience that no stable formulation on acceptable vehiles could be produced as shown in the referenced study.

Test material

Test material information

Constituent 1

Reference substance name:

Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

EC Number:

276-657-4

EC Name:

Hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

Cas Number:

72428-99-0

Molecular formula:

C32H13CuN8O4S2.(SO3H)m.(C4H11NO)n.(C8H18N) (3-m-n).C15H17N3

IUPAC Name:

hydrogen [[[(2-ethylhexyl)amino]sulphonyl][[(3-methoxypropyl)amino]sulphonyl]-29H,31H-phthalocyaninesulphonato(3-)-N29,N30,N31,N32]cuprate(1-), compound with N,N'-di(o-tolyl)guanidine (1:1)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: HARLAN, Kreuzelweg 53,5961 NM HORST- The Netherlands
- Age at study initiation: 3 or 4 weeks old
- Weight at study initiation: 250 - 310 g
- Diet (e.g. ad libitum): SAFE, 106, ad libitum
- Water (e.g. ad libitum): drinking water (tap water from public distribution system), ad libitum
- Acclimation period: a minimum acclimatization period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least ten
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Physiological saline for the intradermal injections and propylene glycol for the topical applications.

Induction 2

Concentration / amount:

Intradermal Induction: 10%, epicutaneous induction: 60%, epicutaneous challange: 30% or 60%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Physiological saline for the intradermal injections and propylene glycol for the topical applications.

Challenge 2

Concentration / amount:

Intradermal Induction: 10%, epicutaneous induction: 60%, epicutaneous challange: 30% or 60%

No. of animals per dose:

GROUP 1 (negative control) : 5
GROUP 2 (treated) : 10

Details on study design:

1st Intradermal Induction:
Day 0
After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:

GROUP 1 (Control):
• 2 ID: Freund's Complete Adjuvant diluted at 50 % in physiological saline
• 2 ID: physiological saline
• 2 ID: a mixture with equal volumes v/v:
- Freund's Complete Adjuvant at 50% and physiological saline

GROUP 2 (Treated):
• 2 ID: Freund's Complete Adjuvant diluted at 50 % in physiological saline
• 2 ID: test item at 10% in physiological saline,
• 2 ID: a test mixture in equal volumes v/v:
- Freund's Complete Adjuvant at 50% and the test item at 20% in physiological saline

2nd Topical Induction:
Day 7
The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick vase line, in order to create a local irritation.

Day 8
A topical application under occlusive dressing for 48 hours was performed on the injection sites of each animal.
GROUP 1 (Control): 0.5 mL of propylene glycol
GROUP 2 (treated): 0.5 mL ofthe test item at 60%

Day 10
Occlusive dressing removal and rinsing with propylene glycol of treated group.

Challenge controls:

Challenge phase
Day 21
The experimental procedure of this phase was identical for both groups GROUP 1 and 2: on the previously shorn dorso-Iumbar zone, an application, under occlusive dressing, was performed during 24 hours:
- 1 sample cup saturated with the test item diluted at 60% and 1 sample cup saturated with the test item diluted at 30%.

Day 22
Occlusive dressing removal.

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:

23 July 2013:

24h reading / Conc.12.5% / 70% of animals sensitized
48h reading / Conc 12.5% / 60% of animals sensitized
24h reading / Conc.6.25% / 70% of animals sensitized
48h reading / Conc 6.25% / 50% of animals sensitized

21 January 2014:

24h reading / Conc.12.5% / 50% of animals sensitized
48h reading / Conc 12.5% / 30% of animals sensitized
72h reading / Conc 12.5% / 30% of animals sensitized
24h reading / Conc.6.25% / 30% of animals sensitized
48h reading / Conc 6.25% / 20% of animals sensitized
72h reading / Conc 6.25% / 70% of animals sensitized

26 August 2014:

24h reading / Conc.12.5% / 80% of animals sensitized
48h reading / Conc 12.5% / 40% of animals sensitized
24h reading / Conc.6.25% / 40% of animals sensitized
48h reading / Conc 6.25% / 0% of animals sensitized

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Challange Concentration 60%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Challange Concentration 60%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Challange Concentration 60%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Challange Concentration 60%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Challange Concentration 30%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Challange Concentration 30%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Challange Concentration 30%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Challange Concentration 30%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Challange Concentration 60%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Challange Concentration 60%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Challange Concentration 60%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Challange Concentration 60%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Challange Concentration 30%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Challange Concentration 30%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Challange Concentration 30%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Challange Concentration 30%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Sodium lauryl sulfate at 10% induced an irritant effect on the skin during the 2nd induction.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In conclusion, under these experimental conditions, 0 % of the test animals showed a sensitisation response. Therefore the test item does not have to be classified as a skin sensitizer in accordance with the Regulation EC No 1272/2008.

Executive summary:

The aim of the study was to evaluate the possible allergenic activity of the test item after intradermal and topical administration in guinea pigs.

After induction (intradermic injection at 10% and topical application at 60%) of 10 Guinea Pigs of treated group with the test item and a 10-day rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 60% and at 30% in propylene glycol. The experimental protocol was established according to the OECD guideline No. 406 dated July 17th , 1992 and the test method B.6 of the council regulation No. 440/2008.

In the treated group (chalange dose of 60%), no cutaneous reaction attributable to allergy was recorded in animals after the challenge phase. In the control group (associated with the challange dose of 60%), no cutaneous intolerance reaction was recorded at 24 and 48 hours. A blue coloration, not preventing erythema quotation was noted at the level of all treated areas at 60%.

In the treated group (challange dose of 30%), no cutaneous reaction attributable to allergy was recorded in animals after the challenge phase. In the control group (associated with the challange dose of 30%), no cutaneous intolerance reaction was recorded at 24 and 48 hours.