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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:
weight of evidence
Study period:
From 15 March 1999 to 20 March 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This non GLP-compliant study was performed with method equivalent to OECD 429 method. However, the test item was used diluted at 0.25, 0.5, 1 and 2% instead of pure recommended by REACh regulation. A sensitisation effect cannot be excluded at higher dose. No test item purity, solubility and stability were provided. No details on test item (purity, batch) and no radiolabelled thymidine information were provided.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The test item was used diluted instead pure required by REACh regulation
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-nitrophenyl)amino]ethanol
EC Number:
225-555-8
EC Name:
2-[(2-nitrophenyl)amino]ethanol
Cas Number:
4926-55-0
Molecular formula:
C8H10N2O3
IUPAC Name:
2-[(2-nitrophenyl)amino]ethanol
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by the Sponsor : Bristol-Myers squibb Worldwide Beauty Care
- Expiration date of the lot/batch: not specified
- Purity test date: not specified

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: not specified
- Specific activity:not specified
- Locations of the label: 3H-thymidine was used in this study
- Expiration date of radiochemical substance: not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test articles will be prepared daily in vehicle at 0.25, 0.50, 1.0 and 2.0% (2wgt/vol)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Jackson laboratories or Harlan Sprague Dawley
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: not specified
- Age at study initiation:8-12 weeks
- Weight at study initiation: 18-25g
- Housing: housed in shoebox-type cages with a filter cover which provides a filtered microenvironment.
- Diet (e.g. ad libitum): Certified Rodent Chow #7012C Teklad, ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 7 days
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 26 deg Celsius
- Humidity (%): 30 to 70%
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 hours / 12 hours
- IN-LIFE DATES: From: 15 March 1999 To: 20 March 1999

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0, 0.25, 0.5, 1 and 2 % were used
No. of animals per dose:
5 females were used per dose
Details on study design:
PRE-SCREEN TESTS:
No pre-screen test was performed before the main study.

MAIN STUDY
Mice were treated on dorsal surfaces of both ears, once per day for 3 days. On day 5 the mice were injected with 20µC of 3H-thymidine in 250 µL. Five hours later the mice were euthanazied with C02 and the draining auricular lymph nodes removed. A single cell suspension was prepared from lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloro-acetic acid (TCA) overnight at 2-8 deg Celsius. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. Incorporation of 3H-thymidine was measured with Beta-scintillation counter.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Increases of 3H-thymidine incorporation relative to vehicle-treated control were derived for each group and recorded as stimulation indices (Test/Control ratio). THe criterion for a positive response is that one or more concentration of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control.

TREATMENT PREPARATION AND ADMINISTRATION:
The test articles will be prepared daily in vehicle at 0.25; 0.5, 1 and 2% (wgt/vol) in vehicle (DMSO). The test articles were applied daily on the dorsal surfaces of both ears for 3 days at a volume of 25 µL.
Positive control substance(s):
other: p-phenylenediamine
Statistics:
Descriptive statistics and an analysis of variance (ANOVA) will be performed. If a statistically significant difference is detected, a Dunnett's test will be performed.

Results and discussion

Positive control results:
The positive control, PPD, at1.0 and 2.0% resulted in test/control ratios greater than 3 (5.06 and 13.79, respectively) indicating a positive response. Only the response at the high dose was statistically significant compared to the vehicle control.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.19
Test group / Remarks:
Test Item at 0.25%
Key result
Parameter:
SI
Value:
0.79
Test group / Remarks:
Test Item at 0.5%
Key result
Parameter:
SI
Value:
1.03
Test group / Remarks:
Test Item at 1.0%
Key result
Parameter:
SI
Value:
1.28
Test group / Remarks:
Test Item at 2.0%

Any other information on results incl. tables

Table 1 :Summary of Results

 

Treatment Group

DPM (mean±sem)

Test/control Ratio

Result

DMSO

0

735±216

 -

 

Test Item

0.25

875±172

1.19

 -

Test Item

0.5

577±137

0.79

 -

Test Item

1

756±376

1.03

 -

Test Item

2

942±146

1.28

 -

PPD

0.25

990±313

1.35

 -

PPD

0.5

1388±281

1.89

 -

PPD

1

3720±663

5.06

 -

PPD

2

10134±4853*

13.79

 +

 

*statistically significant difference (p>0.001) compared to vehicle control

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Under the experimental conditions of this study, the test item HC Yellow No. 2 did not induced delayed contact hypersensitivity to mice after skin contact. However, the study cannot be used for classification because the test item was used diluted at 0.25, 0.5, 1 and 2% instead of pure recommended by REACh regulation. A sensitisation effect cannot be excluded at higher dose. No test item purity, solubility and stability were provided. No details on test item (purity, batch) and radiolabelled thymidine information were provided.
Executive summary:

The purpose of this no GLP compliant study was to evaluate the potential sensitising effect of the test substance HC Yellow No. 2 in mice on a Local Lymph Node Assay (LLNA) which was performed with a method equivalent to OECD Guideline 429 method.

25 μL test preparation applied to dorsal aspect of ear lobe of CBA/Ca mice daily for 3 days. 5 days after the first application, 250 μL of a solution containing 20 μCi/mL 3H-thymidine was injected intravenously. 5 hours later the animals were euthanized and the draining auricular lymph nodes were removed. Incorporation of 3H-thymidine was measured by beta-scintillation counting. The compound is not positive (>3) at any concentration and it is not sensitizer in this test.

Under the experimental conditions of this study, the test item HC Yellow No. 2 did not induced delayed contact hypersensitivity to mice after skin contact. However, the study cannot be used for classification because the test item was used diluted at 0.25, 0.5, 1 and 2% instead of pure recommended by REACh regulation. A sensitisation effect cannot be excluded at higher dose.No test item purity, solubility and stability were provided. No details on test item (purity, batch) and radiolabelled thymidine information were provided.