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Administrative data

Description of key information

In a LLNA-study Stimulation Indices of 1.29, 1.33, and 1.37 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The substance is regarded as not sensitising to the skin.

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
Study period:
2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
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: 9 - 10 weeks
- Weight at study initiation: 18.5 - 20.5 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:
propylene glycol
Concentration:
5, 10, and 25% (w/w)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:

A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used was a 25% suspension in PG. Grinding of the test item in a mortar was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved by the use of other vehicles.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Redness of the ear skin could not be detected, due to the blue colour of the test item.
Thus, the test item in the main study was assayed at 5, 10, and 25% (w/w). 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 (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in PG. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
3.9.2 Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.1 µCi of 3H-methyl thymidine (equivalent to 80.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Determination of incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in 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 scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute.

Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance.

Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser 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 April 2015 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.93, 2.65, and 9.48, respectively.
The EC3 value calculated was 10.8 % (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: PG

 

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

13

---

---

---

---

---

BG II

12

---

---

---

---

0

1

3082

3069.5

8

383.7

1.00

5

2

3984

3971.5

8

496.4

1.29

10

3

4103

4090.5

8

511.3

1.33

25

4

4221

4208.5

8

526.1

1.37

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 all S.I.´s are below the threshold value of 3.

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.A possible erythema of the ear skin could not be detected, due to the blue colour of the test item.

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.

Ear Weights

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A relevant increase in ear weights was not observed.
Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item Fat Blue B 01 was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of Fat Blue B 01, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/w) in PG by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be technically achieved. A control group of four mice was treated with the vehicle (PG) 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. Redness of the ear skin could not be detected, due to the blue colour of the test item.

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 1.29, 1.33, and 1.37 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

not classified

The substance was found not sensitising in an LLNA in mice.