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Toxicological information

Skin sensitisation

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Administrative data

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:
other: Guideline-conform study under GLP without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: Pigment Orange 5
Other name: 1-[(2,4-Dinitrophenyl)azo]-2-naphthol
C.I. Name: Pigment Orange 5 (PO 5)
CAS No.: 3468-63-1
Batch: 6963A
Content (C.I.): 97.8% (w/w)
Certificate of Analysis: AZ 784A/Toxdl, dated: September 18, 2012
Expiry Date: September 06, 2022
Storage Conditions: At room temperature
Stability in Solvent: >72 h in 1,2-propylene glycol at room temperature >72 h in DMSO at room temperature
Safety Precautions: Standard Personal Protection Equipment Avoid dust formation Avoid sources of ignition

In vivo test system

Test animals

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 - 11 weeks
- Weight at study initiation: 19.2 - 24.5 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)

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
2.5%, 5% and 10% (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 could be technically used was a 10% suspension in propylene glycol. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that does at the same time not induce signs of systemic toxicity, a non-GLP irritation pre-test was performed in two animals. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5% and 10% (w/w) 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. The measured ear weight was also considered in this evaluation.
There were no signs of systemic toxicity or excessive local skin irritation. Eventual erythema formation could not be assessed due to the colour of the test item, but increase in ear thickness and ear weights were below the threshold value of ≥25% increase compared to pre-treatment values (for ear thickness) and historical vehicle values (for ear weights), respectively. For further details of this irritation pre-test, see Appendix 1 of the report.
The test item in the main study was therefore assayed at the concentrations 2.5%, 5% and 10% (w/w). The highest concentration tested was the highest level that can be achieved whilst avoiding systemic toxicity and excessive local irritation as confirmed in the pre-experiment described above. The subsequent concentrations correspond to the concentrations specified by the OECD Guideline 429.


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 2.5%, 5% and 10% (w/w) in propylene glycol. 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 after sacrifice; biopsy punches were taken from each ear.

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

Results and discussion

Positive control results:
Experiment performed in October 2012 (Harlan study number 1510201) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 1.5, 1.9, and 5.7, respectively.
The EC3 value calculated was 14.3 % (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.

In vivo (LLNA)

Results
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see depicted table below

Any other information on results incl. tables

Calculation and results of individual data

Vehicle: propylene glycol

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

19

---

---

---

---

---

BG II

19

---

---

---

---

0

1

1967

1948

8

243.5

1.0

2.5

2

2519

2500

8

312.5

1.3

5

3

1314

1295

8

161.9

0.7

10

4

1831

1812

8

226.5

0.9

 

BG=  Background (1 ml 5% trichloroacetic acid) in duplicate

1    =  Control Group

2-4=  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 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 systemic findings were observed during the study period. Eventual erythema of the ear skin could not be evaluated due to the colour of the test item. From days 2 to 5, the feces of the animals had an orange staining due to oral ingestion in the course of grooming activities.

Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Pigment Orange 5 was not a skin sensitiser under the test conditions of this study
Executive summary:

In order to study a possible skin sensitising potential of Pigment Orange 5, three groups each of four female mice were treated once daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in propylene glycol by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (propylene glycol) 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 of 3H-methyl thymidine measured in a beta-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed. Eventual erythema of the ear skin could not be evaluated due to the 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 of 3HTdR 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 1.3, 0.7 and 0.9 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in propylene glycol. A dose response was not observed.

The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.