2-(4-amino-2-nitroanilino)ethanol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 July 2001 to 7 November 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No pre screen test was perform in order to determine the test dose. Control and Test groups were composed by 3 animals. The current OECD Guideline required a minimum of 4 animals per dose group.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

Experimental groups of 3 animals were used instead of 4 required by OECD method

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:

2-(4-amino-2-nitroanilino)ethanol

EC Number:

220-701-7

EC Name:

2-(4-amino-2-nitroanilino)ethanol

Cas Number:

2871-01-4

Molecular formula:

C8H11N3O3

IUPAC Name:

2-(4-amino-2-nitroanilino)ethanol

Test material form:

solid: particulate/powder

Remarks:

Green/Brown crystalline powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplied by Unilever, Lot L-28231
- Expiration date of the lot/batch: not specifed
- Purity test date: 18 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the original containers at room temperature (17-20°C) in the dark
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: samples were stable during 9 month under ambient conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item and vehicle (acetone:olive oil, 4:1 (v/v)) were placed into a glass beaker on a tared Mettler balance and weight/volume dilutions were prepared. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made immediately prior to dosing.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan (Netherlands)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: not specified
- Age at study initiation: 7-12 weeks
- Weight at study initiation: 12.6-25.4g
- Housing:In groups of three in Makrolon type-3 cages with standard softwood bedding
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433 ad libitum
- Water (e.g. ad libitum):Community tap water from Itingen available ad libitum
- Acclimation period: 6 days of acclimation
- Indication of any skin lesions: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70% relative humidity
- Air changes (per hr): 10-15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period
- IN-LIFE DATES: From: 10 July 2010 To: 25 July 2010

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0, 0.10, 0.25, 0.50, 1.00, 2.50% (w/v)

No. of animals per dose:

3 animals were used per dose

Details on study design:

PRE-SCREEN TESTS:
No screen test were performed

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Animals were assignated randomly by computer algorithm
- Criteria used to consider a positive response:
A test item is regarded as sensitizer in a LLNA if the following criterias are fulfilled
-That exposure to at least one concentration of the test item resulted in an incorporation of radiolabelled thymidine at least 3 fold or greater than that recorded in control miice, as indicated by the stimulation index
-That the data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression

TREATMENT PREPARATION AND ADMINISTRATION: The preparations of the test item were made immediately prior each dosing. Mice were treated by (epidermal) topical application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of in acetone:olive oil 4:1. The application volume, 25µL, was spread over the entire dorsal surface of each ear lobe once daily for three consecutive days. A further group of mice were treated with an equivalent volume of the relevant vehicle alone (control animal). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.

Five days after topical application, the mice were administered with 250µL of radiolabelled thymidine (specific activity 2Ci/mmol ; concentration 1 mCi/mL) by intravenous injection via a tail vein. Five hours later, mice were euthanized.The draining lymph node were excised and pooled for each experimental group. They were washed and resuspended with trichloroacetic acid (at 5%) and incubated overnight at 4°C. The precipitates were resuspended in trichloroacetic acid (at 5%) and transferred to glass scintilation vials in order to count the incorportation of radiolabelled thymidine by Beta-scintillation counter.

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:

Stimulation indices of 2.4, 3.7 and 7.0 were determined at a concentration of 5, 10 and 25% of positive control in acetone:olive oil 4:1 (v/v). It induced sensitising effects when tested at concentrations of 10% and 25%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.5

Test group / Remarks:

0.10%

Results 2

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

0.25%

Results 3

Key result

Parameter:

SI

Value:

1.9

Test group / Remarks:

0.50%

Results 4

Key result

Parameter:

SI

Value:

1.8

Test group / Remarks:

1.00%

Results 5

Key result

Parameter:

SI

Value:

3.3

Test group / Remarks:

2.50%

Results 6

Key result

Parameter:

other: dpm per lymph node

Value:

467

Test group / Remarks:

Vehicle control group

Cellular proliferation data / Observations:

No evidence of stimulating allergic contact sensitisation when HC Red n° 3 was tested at 0.1%, 0.25%, 0.5% and 1.0% (w/v) as the mean stimulation index did not exceed a value of 3, but HC Red n° 3 showed a positive lymphocyte proliferative increase when tested at 2.5% (w/v).

Any other information on results incl. tables

Table 1 :Summary of the results

Concentration % (w/v)	Mean Stimulation Index (SI)
	HC Red n° 3	Alpha Hexylcinnamaldehyde
0.1	0.5	-
0.25	1.2	-
0.5	1.9	-
1.0	1.8	-
2.5	3.3	-
5.0	-	2.4
10.0	-	3.7
20.0	-	7.0

 

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Under the experimental condition of the study, the HC Red No. 3 at the concentration of 2.5% induced contact allergenic effect when administered to the dorsum of ear lobes in mice. HC Red n° 3 was sensitising when tested at 2.5%. Hence the test item was classified as sensitizer Category 1A strong sensitiser according to the CLP criterias. However, this study had some deviations as the insufficient number of animals per group (3 were used instead of 4 required in the OECD guideline) but the results were still relevant.

Executive summary:

The purpose of this GLP compliant study was to identify the contact allergenic potential of the test item HC Red No. 3 when applied to the dorsum of both ear lobes of mice in a LLNA test performed according to OECD 429 method.

Concentrations of 0, 0.1, 0.25, 0.5, 1.0 and 2.5% HC Red 3 (w/v) in acetone:olive oil, 4:1 (v/v) applied topically to the dorsum of each ear lobe of CBA/CaOlaHsd mice (left and right) (25 µl per ear) once daily on 3 consecutive days.

On day 5, mice were injected intravenously with 250 µl of 3H-methyl thymidine (21.44 µCi). Five hours later (six hours with positive control), mice were euthanized and the draining auricular lymph nodes removed. Lymph nodes from each animal within a dose group were pooled and a cell suspension prepared. After three phosphate buffered saline washes, cells were precipitated in 3 ml of 5% trichloroacetic acid (TCA) at 4°C overnight. Samples were resuspended in 1 ml of 5% TCA and transferred to scintillation vials containing 10 ml of scintillation fluid and analysed.

No evidence of stimulating allergic contact sensitisation when HC Red n° 3 was tested at 0.1%, 0.25%, 0.5% and 1.0% (w/v) as the mean stimulation index did not exceed a value of 3, but HC Red n° 3 showed a positive lymphocyte proliferative increase when tested at 2.5% (w/v).

Under the experimental condition of the study, the HC Red No. 3 at the concentration of 2.5% induced contact allergenic effect when administered to the dorsum of ear lobes in mice. HC Red n° 3 was sensitising when tested at 2.5%. Hence the test item was classified as sensitizer Category 1A strong sensitizer according to CLP criterias. However, this study had some deviations as the insufficient number of animals per group (3 were used instead of 4 required in the OECD guideline) but the results were still relevant.