[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Remarks:

LLNA study has been conducted since in vitro/ in chemico models show conflicting results.

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Molecular formula:

Cannot be adequately assigned

IUPAC Name:

copper(II) 3,3',3'',3'''-(29H,31H-phthalocyanine-2(or 3),9(or 10),16(or 17),23(or 24)-tetrayltetrasulfonyl)tetrapropane-1-sulfonate and its amides with (2-hydroxypropyl)amine, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Molecular formula: C50H52CuN10Na2O20S8 (main component)
Molecular weight: 1479.02 (main component)
Purity of test item: 93.1%
Impurity
Water: 6.5%
EDTA-4Na: 0.4%
Supplier: FUJIFILM Corporation
Lot number: M-002P
Storage conditions: Store in the dark at room temperature.

Specific details on test material used for the study:

Not specified.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Dose Range Finding Animals
Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during
the test: Good conventional
Justification of strain: Same strain is used in the Dose Range Finding test (DRF) and
in the main test.
Number of animals: 8 animals (2 animals/groups)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice,
8 weeks old at start of the DRF
Acclimatization time: 14 days prior to the DRF

Main Test Animals
Species and strain: CBA/Ca Ola Hsd mice
Source: TOXI-COOP ZRT.
H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during
the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories,
mice of the CBA/Ca strain were found to exhibit a more marked
response than other strains. Females are used because the
existing database is predominantly based on females.
Number of animals: 28 animals*/main test (4 animals/treatment group)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice;
9, 10 or 12 weeks old (at start of the main test)
Body weight range
at starting: 18.8 – 23.9 g
The weight variation in animals involved in the study did not
exceed ± 20 % of the mean weight.
Acclimatization time: 14 days

* Including control animals shared with the concurrent LLNAs performed at the same date.
Number of shared animals: 8.

Husbandry
Animal health: Only healthy animals (and not showing any sign of skin
lesion) were used.
Housing during
acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with
deep wood sawdust bedding, to allow digging and other normal
rodent activities.
The same conditions were used for the dose range finding and main test animals. There were
no deviations from these specifications during the experimental phases. The temperature and
relative humidity were recorded daily during the acclimatization and experimental phases.
Before housing the animals, the microbiological status of the room was checked.

Food and Water Supply
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by
ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum.
The food is periodically analysed and is considered not to contain any contaminants that could
reasonably be expected to affect the purpose or integrity of the study. Copies of the relevant
Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive. Relevant batch
number, expiry date and contents of the diet are given in Appendix III.
Animals received tap water from watering bottles ad libitum. The drinking water is
periodically analysed and is considered not to contain any contaminants that could reasonably
be expected to affect the purpose or integrity of the study. Copies of the relevant Certificates
of Analysis are maintained in TOXI-COOP ZRT.’s archive.

Bedding
Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG
(D-73494 Rosenberg (Germany) Holzmühle 1) was available to animals during the study.
Copies of the relevant Certificates of Analysis are maintained in TOXI-COOP ZRT.’s archive.

Identification of Animals
The individual identification of the animals was performed by numbers on the tail. The cages
were marked with identification cards, with information (at least) about study code, species,
strain, sex, dose group and individual animal numbers.

Randomization
The animals were set in order of their body weight. The animals were randomly assigned to
control and test groups using a randomization scheme. The randomization was checked by
computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the
homogeneity and deviations between the groups.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Each mouse was topically treated with 25 μL of 25%, 10%, 5% and 2.5% w/v of test item.

No. of animals per dose:

4 animals per dose

Details on study design:

Dose Range Finding Test
The pre-experiment on formulation evaluation and the dose range finding test (DRF) were not
performed in compliance with the GLP-Regulations and are excluded from the Statement of
the Study Director, but the raw data of these tests are archived under the study code of the
present study.
The maximum dose selection was performed according to the relevant guidelines. The
preliminary irritation/toxicity screen was conducted in a similar experimental manner to the
exposure phase of the main test except there was no assessment of lymph node proliferation
and fewer animals were used.
The test item was formulated in DMSO and evaluated at concentrations of 25 %, 10 %, 5 %
and 2.5 % (w/v) in the DRF. All formulations (apparently solutions) were adequately
applicable on the ears of animals. Groups of 2 CBA/Ca mice were treated with the appropriate
formulations once daily for 3 consecutive days. All animals were observed for any clinical
signs of systemic toxicity or local irritation at the application site during the test. Body weights
were recorded prior to the first treatment (on Day 1) and prior to termination (on Day 6). Both
ears of each mouse were observed for erythema and scored. Measurement of ear thickness was taken using digital micrometer on Day 1 (pre-dose), Day 3 (prior to the treatment) and Day 6.
No mortality, significant, treatment related effect on body weights or any other sign of
systemic toxicity were observed. No sign of significant irritation (indicated by an erythema
score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement).
It should be noted that intensive colour of the test item formulations (blue) made observation
of erythema difficult on the treatment days (after the treatment), but it is considered that a
severe effect (an erythema scored as ≥ 3) would have been visible. No other local
effect was observed in any dose group during the test.
Based on this the 25 % (w/v) concentration (based on solubility) was used as the maximum
in the main test with the aim of testing the highest possible concentration. The test item was
tested also at three additional, lower concentrations (10 %, 5 % and 2.5 %, w/v) to evaluate
dose-response relationship and ensure validity of the test in accordance with the relevant
guidelines.

Main Test Design
The test item was administered at four different concentrations.

In vivo Treatment
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item,
the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear.
After the treatment animals were returned to their cages. Each animal was dosed once a day
for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation toxicity
(significant loss of body weights or other significant clinical symptoms) or excessive skin
irritation (erythema scored as ≥ 3) and no technical treatment failures were observed during
the test. All animals treated were processed and therefore no treatment group was excluded
from the evaluation.

Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS
(1 x PBS, diluted from 10x concentrate) containing approximately 20 μCi# of
3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected,
mice were left for 5 hours (± 30 minutes).
# Remark: Breaking down of [methyl-3H]-Thymidine in aqueous solution (initially approximately 5 % per month; stability is non-linear) is taken into account as necessary when 3HTdR solution is prepared.

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical
dislocation. The draining auricular lymph nodes were excised by making a small incision in
the skin between the jaw and the sternum, pulling the skin gently back towards the ears and
exposing the lymph nodes. Then the nodes were removed using forceps.
Once removed, the nodes of the mice from each test group were pooled and collected
separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet
before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was
prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph
nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was
washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF)
of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was
removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated
before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure
was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of
supernatant above each pellet. The pellets were gently agitated before suspending the LNCs
in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation
of the macromolecules. After incubation with 5 % TCA at 2-8°C overnight (approx. 18 hrs),
each precipitate was removed by centrifugation of the samples at approximately 190 x g for
10 minutes at 4°C and decanting the supernatants. Then the pellets were re-suspended in 1 mL
of 5 % TCA, dispersed using an ultrasonic water bath and stored at 2-8°C until measurement.
On the day of the measurement sample were dispersed using an ultrasonic water bath,
transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently
mixed and loaded into the β-scintillation counter. 3HTdR incorporation was measured for up
to 10 minutes per sample.
The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration
per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots
of 5 % TCA. Instrument used for the measurement:

Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated
in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of
toxicity were observed in the positive control group.
A significant lymphoproliferative response (SI ≥ 3) was noted for HCA (SI = 14.0). The
results of the positive control item demonstrated an appropriate performance of the test in
accordance with the relevant guidelines and confirmed the validity of the assay.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

Dye-2019, 2.5% in DMSO

Results 2

Key result

Parameter:

SI

Value:

3

Test group / Remarks:

Dye-2019, 5% in DMSO

Results 3

Key result

Parameter:

SI

Value:

3.1

Test group / Remarks:

Dye-2019, 10% in DMSO

Results 4

Key result

Parameter:

SI

Value:

2.3

Test group / Remarks:

Dye-2019, 25% in DMSO

Results 5

Key result

Parameter:

other: Erythema Scores

Value:

0

Test group / Remarks:

Dye-2019, 2.5% in DMSO Intensive blue colour of the test item made observation of the erythema difficult. It was considered that a sever effect (an erythema scored as ≥ 3) would have been visible.)

Results 6

Key result

Parameter:

other: Erythema Scores

Value:

0

Test group / Remarks:

Dye-2019, 5% in DMSO Intensive blue colour of the test item made observation of the erythema difficult. It was considered that a sever effect (an erythema scored as ≥ 3) would have been visible.)

Results 7

Key result

Parameter:

other: Erythema Scores

Value:

0

Test group / Remarks:

Dye-2019, 10% in DMSO Intensive blue colour of the test item made observation of the erythema difficult. It was considered that a sever effect (an erythema scored as ≥ 3) would have been visible.)

Results 8

Key result

Parameter:

other: Erythema Scores

Value:

0

Test group / Remarks:

Dye-2019, 25% in DMSO Intensive blue colour of the test item made observation of the erythema difficult. It was considered that a sever effect (an erythema scored as ≥ 3) would have been visible.)

Cellular proliferation data / Observations:

The maximum dose selection was based on results of the formulation evaluation and the dose
range finding test (DRF) according to the relevant guidelines. The maximum
concentration producing adequate homogeneous formulation (apparently solution) suitable
for application on the dorsum of ears of animals was 25 % (w/v) in Dimethyl sulfoxide
(DMSO). No higher test concentration was achievable.
As no significant adverse effects (systemic toxicity or irritation) were observed in the DRF
up to this maximum concentration the test item was examined in the main test as 25 %, 10 %,
5 % and 2.5 % (w/v) formulations in DMSO.
Based on the positive control result the test was valid. No confounding effects of irritation or
systemic toxicity were observed during the main test. According to this the proliferation
values obtained are considered to reflect the real potential of the test item to cause/not cause
lymphoproliferation in the Local Lymph Node Assay.
Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant
control (DMSO) was noted for Dye-2019 at test concentrations of 10 % and 5 % (w/v) being
just on the borderline of SI = 3. No significant effect on the lymphoproliferation was observed
in the other dose groups, however increased values (compared to the control) were observed
at all tested concentrations. The observed SI values were 2.3, 3.1, 3.0 and 2.3 at test item
concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No monotonic linear
dose-response curve was observed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Dye-2019 tested at the maximum feasible concentration of 25 % (w/v, based on solubility) and also at concentrations of 10 %, 5 % or 2.5 % (w/v) as adequate homogeneous formulations [solutions, prepared with Dimethyl sulfoxide (DMSO) as vehicle] was shown to have no skin sensitization potential in the Local Lymph Node Assay.

Executive summary:

The aim of this study was to evaluate the skin sensitization potential of the test item Dye-2019 following dermal exposure in the Local Lymph Node Assay.
Preliminary tests [formulation evaluation and a dose range finding test (DRF)] were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. The maximum concentration producing adequate homogeneous formulation (apparently solution) suitable for application on the dorsum of ears of animals was 25 % (w/v) in Dimethyl sulfoxide (DMSO). No significant adverse effects (systemic toxicity or irritation) were observed in the DRF up to this maximum concentration. According to this the test item was examined in the main test at concentrations of 25 %, 10 %, 5 % and
2.5 % (w/v).
An appropriate positive control (α-Hexylcinnamaldehyde, HCA), and furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed. The positive control item [25 % (w/v) HCA in Acetone: Olive oil 4:1 (v/v) mixture (AOO)] induced significant stimulation over the relevant control (SI = 14.0) thus confirming the
validity of the assay.
No mortality or signs of systemic toxicity were observed during the main test. No significant, treatment related effect on the body weights was observed in any test group. No signs of significant irritation or any other local effects were observed at the treatment site (ears) during the test. It should be noted that intensive colour of the test item formulations (blue) made observation of erythema difficult, but it is considered that a confounding, severe effect would have been visible.
Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMSO) was noted for Dye-2019 at test concentrations of 10 % and 5 % (w/v) being just on the borderline of SI = 3. No significant effect on the lymphoproliferation was observed in the other dose groups, however increased values (compared to the control) were observed at all tested concentrations. The observed SI values were 2.3, 3.1, 3.0 and 2.3 at test item
concentrations of 25 %, 10 %, 5 % and 2.5 % (w/v), respectively. No monotonic
dose-response curve was observed (p = 0.66, r2 = 0.11, evaluated by linear regression using the obtained SI values).
It is known that real sensitisation effect has strong linear dose-response correlation. Based on this, it is considered that the observed increased lymphoproliferation is not caused by sensitization but by some other mechanism.
According to this and in accordance with the relevant test guidelines, the lack of a significantly increased lymphoproliferation at the maximum achievable concentration of 25 % (w/v, based on solubility) and the lack of a significant dose-response relationship together are considered as evidence that Dye-2019 is not a skin sensitizer.
In conclusion, under the conditions of the present assay, Dye-2019 tested at the
maximum achievable concentration of 25 % (w/v; based on solubility) and also at
concentrations of 10 %, 5 % or 2.5 % (w/v) as adequate homogeneous formulations [solutions, prepared with Dimethyl sulfoxide (DMSO) as vehicle] was shown to have no skin sensitization potential in the Local Lymph Node Assay.