Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-07 to 2018-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

May 30, 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

EC Number:

284-943-5

EC Name:

Bis(N,N-dimethylpropane-1,3-diamine-N)[29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]cobalt(1+) chloride

Cas Number:

84989-53-7

Molecular formula:

C42H44CoN12.Cl

IUPAC Name:

hydroxylamine

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Animals:
- Source:Harlan
Sex: Female (nulliparous and non-pregnant)
Age at the beginning of the study: 8-9 weeks
The animals were derived from a controlled full-barrier maintained breeding system (SPF).

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH value of approx. 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Adequate acclimatisation period (at least five days) under laboratory conditions

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

6.25%, 12.5% and 25% (w/v)

No. of animals per dose:

Number of animals: 5 mice / group
5 mice / pre-screen test

Details on study design:

Preparation of the Animals:
- The animals were randomly selected using the validated departmental computerised system
- Identification was ensured by cage number and individual marking (tail)

Pre-screen Test:
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation.

Controls:
AOO (acetone/olive oil) was used as vehicle and served as negative control.

Clinical Observation:
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application. The observation period lasted until study day 6 on which the animals were sacrificed.

Weight Assessment:
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested.

Test Regime:
- Topical application of the test item: Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

- Administration of 3H-Methyl Thymidine:
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

- Preparation of Cell Suspension:
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, weighed, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

- Determination of Incorporated 3H -Methyl Thymidine:
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

- Evaluation of Results:
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.

A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).

Positive control substance(s):

other: 1% phenylenediamine in AOO

Statistics:

not specified

Results and discussion

Positive control results:

A positive control was performed concomitantly.
The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

23.7

Variability:

SD = 3.8

Test group / Remarks:

concentration 6.25%

Results 2

Key result

Parameter:

SI

Value:

23.8

Variability:

SD = 11.0

Test group / Remarks:

concentration 12.5%

Results 3

Key result

Parameter:

SI

Value:

12

Variability:

SD = 3.0

Test group / Remarks:

concentration 25.0%

Any other information on results incl. tables

Pre-screen and solubility results:

The pre-screen test was performed with test concentrations 50 and 25%. After evaluation of the results the maximum applicable and accurate concentration in the vehicle was found to be 25%. Neither signs of systemic toxicity nor signs of excessive irritation were observed. Clinical observations: black discouloured faces and sticky fur. Based on the pre-screen results, following test concentrations were selected 6.25%, 12.5% and 25%.

Main test:

All of the three tested concentrations exceeded the stimulation index of 3:

23.7 at 6.25 %

23.8 at 12.5%

12.0 at 25%

EC3 value could not be calculated since all 3 results were above 3. Sub-categorization in category 1A or 1B is not possible.

- Body Weight Development:

All animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the study.

- Clinical Observation:

All animals survived throughout the test period showing clinical signs. The most relevant findings were black discoloured application sites, black discoloured faeces, and stick fur.

- Weight of Lymph Nodes:

The means of the lymph node weights per group showed no relevant difference compared to the negative control.

The mean weight of the lymph nodes

for the 6.25% test group was                  3.7 mg

for the 12.5% test group was                  3.7 mg

for the 25.0% test group was                  3.8 mg

for the negative-control group was        1.5 mg

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

All of the tested concentrations exceeded the stimulation index of 3. The test item was hence found to be a skin sensitiser in the LLNA when tested at 6.25, 12.5 and 25.0% (v/v) in acetone:olive oil 4:1. The EC3 value could not be calculated as the stimulation indices of all concentrations were above 3.

Executive summary:

In order to study the skin sensitization potential of the test item a local lymphe node assay was conducted according to OECD 429 and under GLP. Three groups each of five female mice were treated daily with the test item at concentrations of 6.25, 12.5 and 25.0 % (v/v) in acetone:olive oil (4:1) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of five mice was treated with the vehicle (AOO) 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 animal. 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 of 3H-methyl thymidine measured in a β-scintillation counter.

Neither mortality nor effects on body weights but clinical signs were observed. The most relevant findings were black discoloured application sites, black discoloured faeces, and stick fur.

In this study Stimulation Indices of 23.7, 23.8 and 12.0 were determined with the test item at concentrations of 6.25, 12.5 and 25% in AOO. The test item is hence found to be skin sensitizer. As all of the tested concentrations exceeded the Stimulation Index of 3, the EC3 value could not be calculated.

The results of the radioactivity determination are supported by the second endpoint, the means of the lymph node weights per group, which showed increased values compared to the negative control values.