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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Jan. 15, 2004 to April 22, 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
according to US FDA, French and OECD principles of GLP
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material: 2-Amino-6-chloro-4-nitrophenol, Chloro orange base (Code# A000119)
- TSIN: WR23214
- Substance type: Pure active substance
- Physical state: Orange powder
- Stability under test conditions: The substance is considered to be stable for more than 7 years if stored dry and protected from light and humidity at room temperature
- Stability in solution: The test substance has showed very good stability when tested in DMSO solution (approximately 10% w/v), acetone/water solution (1:1, 7% w/v) and water solution (approximately 0.05% w/v) over a period of 7 days
- Solubility: Solubility in different solvents is as follows:
0.07-0.24 % (pH 4.3) in water
8.7 weight% (pH 3.6) in acetone/water 1:1
> 10 weight% in DMSO
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: n°Batch :Wella AG ; L-19503
- Expiration date of the lot/batch: 8 May 2005
- Purity test date: No data
- Purity : 99.9% (HPLC at 254 nm)

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: No data
- Specific activity:23.3 µCi
- Locations of the label: [3H] methyl thymidine
- Expiration date of radiochemical substance: No data

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light and humidity.
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/vehicle: No data

TREATMENT OF TEST MATERIAL PRIOR TO TESTING : No

In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CBA/J mouse were obtained from Centre d'Elevage Janvier, route des Chenes Secs, 53940 Le Genest Saint Isle, France
- Age at study initiation: Approximately 10 weeks
- Weight at study initiation: 19 to 25 g
- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x 160 x 140 mm). Bedding composed of dust-free sawdust made from spruce tree wood was analyzed at least twice a year for chemical and bacterial contaminants.
- Diet: Mouse pelleted complete diet; ad libitum
- Water: Softened and filtered (0.2µm) mains drinking water; ad libitum
No known contaminants were present in bedding, diet or water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: 10 days between animal arrival and the start of treatment. All animals received a clinical inspection for ill-health on arrival. Allocation to treatment groups was performed during the acclimatization period, using a computer general randomization.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: >40% (target range)
- Air changes: Minimum 8 air changes per hour
- Photoperiod: 12 hours light (artificial)/12 hours dark

IN-LIFE DATES: From: Jan. 23, 2004 To: Jan. 28, 2004

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Remarks:
(vehicle 1) and acetone/water (1:1) mixed with olive oil (75%/25%) (vehicle 2)
Concentration:
0.5, 1.5. 5.0 and 10.0% concentrations of test substance tested in each vehicle 1 and vehicle 2.
No. of animals per dose:
5 female mice per group
Details on study design:
RANGE FINDING TESTS: No range-finding study was performed. The test concentrations of the study were chosen by the Study Sponsor based on maximum solubility.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay for 2-Amino-6-chloro-4-nitrophenol
- Allocation to treatment groups : performed during the acclimatisation period, using a computer general randomisation.
- Criteria used to consider a positive response: A test substance was considered to have skin sensitizing activity if, at one or more concentrations, it induces 3-fold or greater increase in isotope incorporation relative to the vehicle. Thus, a stimulation index ≥ 3.0 was regarded as a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
A) Test substance preparation: The test substance was prepared daily as a solution in DMSO and acetone/water (1:1) mixed with olive oil at concentrations of 0.5, 1.5, 5.0 and 10.0%. The test preparations were used within 5 hours of preparation.
B) Administration of the test preparations: On Day 0, 1 and 2, 25 µl of the test substance formulation or vehicles was applied on the dorsal surface of each ear. A hair dryer was used for approximately 5 minutes to dry the application sites.

OBSERVATIONS:
- Morbidity/mortality: All animals were observed at least daily.
- Clinical signs: On treatment days, animals were examined before and at least once after dosing to detect any clinical signs or reaction to treatment
- Body weights: All animals were weighed on Days -1 and 5.
- Evaluation of cell proliferation: On Day 5 the mice were injected, i.v., with 250 μL tested in each vehicle 1 and vehicle 2 of phosphate buffered saline (PBS) containing 23.3 μCi of [3H] methyl thymidine. Five hours later the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph node taken and weighed. A single cell suspension was prepared for each animal. Cells were washed twice with PBS and precipitated with ice cold 5% trichloro-acetic acid (TCA). 18 hours later the pellets were resuspended in 1 mL of TCA and transferred into the scintillation cocktail. 3H-methyl thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter (Packard).

Details on the counting system were provided in the study report.
Positive control substance(s):
other: 1% p-phenylenediamine (PPD) (The positive control group was included in another study (study no. 762/040) and was common to this study)
Statistics:
No statistical analysis were performed

Results and discussion

Positive control results:
- Mean DPM: 2771 ± 1614
- Mean Stimulation index: 7.0 (The SI is greater than 3 thus validated the experimental conditions of OECD 429)
- EC3 value for the positive control was not calculated (one single value).

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
EC3
Value:
ca. 6.85
Test group / Remarks:
in DMSO
Key result
Parameter:
EC3
Value:
ca. 0.68
Test group / Remarks:
in acetone/water/olive oil
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test item with DMSO at 0.5%
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
Test item with DMSO at 1.5%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
Test item with DMSO at 5.0%
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
Test item with DMSO at 10.0%
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
Test item with Acetone/water/olive oil at 0.5%
Key result
Parameter:
SI
Value:
3.9
Test group / Remarks:
Test item with Acetone/water/olive oil at 1.5%
Key result
Parameter:
SI
Value:
3.7
Test group / Remarks:
Test item with Acetone/water/olive oil at 5.0%
Key result
Parameter:
SI
Value:
5.2
Test group / Remarks:
Test item with Acetone/water/olive oil at 10%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
- In DMSO, mean stimulation indices for 2-Amino-6-Chloro-4-Nitrophenol WR 23214 at the concentrations of 0.5%, 1.5%, 5.0% and 10.0% were 1.2, 1.2, 2.0 and 4.7 respectively. The response was thus considered to be positive.
- In acetone/water/olive oil, mean stimulation indices for 2-Amino-6-Chloro-4-Nitrophenol WR 23214 at the concentrations of 0.5%, 1.5%, 5.0% and 10.0% were 2.8, 3.9, 3.7 and 5.2 respectively.

EC3 CALCULATION
- EC3 value was not calculated for the positive control p-phenylenediamine (PPD) (one
single value).
- EC3 value calculated for 2-Amino-6-Chloro-4-Nitrophenol WR 23214 was 6.85% in
DMSO and 0.68% in acetone/water/olive oil

CLINICAL OBSERVATIONS:
- Mortality:No animal died during the study
- Clinical Signs: No clinical signs were observed in the treated animals during the study

BODY WEIGHTS
- Minor body weight loss (less than 10%) in few animals was observed which was considered to be purely coincidental.

Any other information on results incl. tables

Evaluation of Lymph Nodes Weight:

- In animals given the positive control p-phenylenediamine (study number 762/040), the ratio of mean lymph node weights (positive control)/mean lymph node weights (vehicle 1) was 1.2 at the concentration of 1%, indicating an immune response.

- In animals given test substance in DMSO, the ratio of mean lymph node weights (test substance)/mean lymph node weights (vehicle 1) was 1.1, 1.2, 1.0 and 1.3 at the concentrations of 0.5, 1.5, 5.0 and 10.0%, respectively. Lymph node weights of the test substance group at the concentrations of 1.5 and 10.0% were therefore considered to indicate an immune response comparable to the positive control.

- In animals given test substance in acetone/water/olive oil, the ratio of mean lymph node weights (test substance)/mean lymph node weights (vehicle 2) was 1.2, 1.1, 1.1 and 1.2 at the concentrations of 0.5%, 1.5%, 5.0% and 10.0% respectively. Lymph node weights of the test substance group at the concentrations of 0.5% and 10% were therefore considered to indicate an immune response comparable to the positive control

Table 1: Summary of the results – mean DPM and stimulation index (Study # 67975)

Test substance

Concentration

Mean DPM

Stimulation index

2-Amino-6-chloro-4-nitrophenol in DMSO

Vehicle DMSO

448

1.0

0.5%

522

1.2

1.5%

534

1.2

5.0%

910

2.0

10.0%

2118

4.7

2-Amino-6-chloro-4-nitrophenol in acetone /water/olive oil

Vehicle Acetone/water/olive oil

166

1.0

0.5%

470

2.8

1.5%

642

3.9

5.0%

608

3.7

10.0%

861

5.2

Positive control PPD in DMSO

1%

2771

7.0

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: expert judgment
Conclusions:
2-Amino-6-chloro-4-nitrophenol was determined to be a skin sensitizer in the Local Lymph Node Assay (LLNA) when tested at concentrations of 0.5, 1.5, 5.0 and 10.0% in DMSO and acetone/water (1:1) mixed with olive oil (4:1).

Executive summary:

The study was performed to assess the skin sensitization potential of 2-Amino-6-chloro-4-nitrophenol by following the OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

Ten week old female CBA/J mice, weighing 19 to 25 g (source Centre d'Elevage Janvier, route des Chenes Secs, 53940 Le Genest Saint Isle, France) were used in the study. Animals were housed in groups of 5 in plastic cages (265 x 160 x 140 mm). Animals were maintained under standard laboratory conditions (temperature: 19 to 25°C, humidity: >40%; minimum 8 air change/hour and 12 hour light/12 hour dark cycle). Prior to the treatment, animals were acclimatized under laboratory conditions for a minimum period of 9 days. The animals received mouse pelleted complete diet and filtered (0.2 µm) mains drinking water ad libitum. All animals received a clinical inspection for ill-health on arrival.

Two vehicles were used for the preparation of test substance, vehicle 1 (dimethyl sulfoxide (DMSO) and vehicle 2 (acetone/water/olive oil). The test substance was prepared daily in vehicle 1 or 2 at concentrations of 0.5%, 1.5%, 5.0% and 10.0%. The test preparations were used within 5 hours of preparation. 1% p-phenylenediamine (PPD) served as the positive control.

 

On Days 0, 1 and 2, the animals received 25 µL of the test substance or vehicle on the dorsal surface of each ear. A hair dryer was used for approximately 5 minute to dry pinnae.

On Day 5 of the study, all mice received an intravenous injection of 250 µL of phosphate buffered saline (PBS) containing 23.3 µCi of [3H] methyl thymidine. Approximately 5 hours later, the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph nodes taken and weighed. A single cell suspension of lymph node cells was prepared for each animal. Cells were processed further and transferred into scintillation cocktail. 3H-methyl thymidine incorporation was measured by liquid scintillation counting in a TRI-CARB 2700TR counter (Packard). All animals were observed for mortality/morbidity and clinical signs. Body weights were recorded on Days 1 and 5.

There was no mortality as well as no treatment-related effects in body weight or body weight gains observed during the study. No treatment-related clinical signs were observed. 

Mean Stimulation index (SI) for the positive control (1% PPD) relative to the vehicle was 7.0 which was greater than 3 thus demonstrating the sensitivity and validity of the test system used.

The mean SIs of the test substance at the concentrations of 0.5, 1.5, 5.0 and 10.0% in DMSO were 1.2, 1.2, 2.0 and 4.7, respectively. The mean SIs at the concentrations of 0.5, 1.5, 5.0 and 10.0% in acetone/water/olive oil were 2.8, 3.9, 3.7 and 5.2 respectively. Thus the test substance elicited a positive response.

EC3 values calculated were 6.85% in DMSO and 0.68% in acetone/water/olive oil. The EC3 value in acetone/water/olive oil of 0.68% was considered to be an underestimation due to the unusually low mean DPM value for the control.

Based on above, 2-Amino-6-chloro-4-nitrophenol was determined to be a skin sensitizer in the Local Lymph Node Assay (LLNA) when tested at concentrations of 0.5, 1.5, 5.0 and 10.0% in DMSO and acetone/water (1:1) mixed with olive oil.

This LLNA study is classified as acceptable, and satisfies the guideline requirements of OECD 429 (Skin Sensitisation: Local Lymph Node Assay).