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Description of key information

The substance was investigated in a GLP compliant local lymph node assay (OECD 429). Three groups each of four female mice were treated daily with Pigment Green 7 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 topical application the mice were injected intravenously intoa 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 of 3H-methyl thymidine measured in a ß­scintillation counter.

No clinical signs were observed in animals. All treated animals survived the scheduled study period.

Calculation of the EC 3 value was not possible because no test concentrations produced a STIMULATION INDEX (S.1.) of 3 or higher. As a result the substance is classified as a non-sensitizer.

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
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
Purity: >95%
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:

In another in vivo study with guinea pigs polychloro copper phthalocyanine apparently gave no evidence of skin sensitization in animal studies according to the authors (Putilina 1976, Val. 4). Relevant details were not reported (i.e. induction/ challenge routes and concentrations).

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 indeces 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 fourteenth time in Regulation (EC) No. 2020/217.