(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From March 10, 1999 to April 20, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

The study was conducted in general compliance with the methodology published in Methods in Immunotoxicology, Vol. 2, pages 279-290, 1975

Deviations:

yes

Remarks:

A single cell suspension was prepared for each sub-group and not for each group. Allocation to treatment groups was not performed at the completion of the acclimatation period but during this period.

GLP compliance:

yes

Remarks:

according to OECD and US FDA principles of GLP

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

EC Number:

280-734-8

EC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Cas Number:

83763-48-8

Molecular formula:

C9H14N2O2.H2O4S

IUPAC Name:

(3-ammonio-4-methoxyphenyl)(2-hydroxyethyl)ammonium sulphate

Constituent 2

Reference substance name:

Lehmann Blau

IUPAC Name:

Lehmann Blau

Constituent 3

Reference substance name:

2-amino-4-hydroxyethylaminoanisole sulfate

IUPAC Name:

2-amino-4-hydroxyethylaminoanisole sulfate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Lehmann Blau (1-methoxy-2-amino-4-β-hydroxyethylaminobenzene-dihydrochloride)
- Substance type: Pure active substance
- Physical state: Grey powder
- Storage condition of test material: At room temperature

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Supplier # Wella AG ; Batch # 57 (Sample No.: R97005275)
- Expiration date of the lot/batch: Not specified
- Purity test date: 8 January 1996

- Composition of test material, percentage of components: 97.7% Lehmann-Blau



STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: grey powder form, at 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
- Treatment of test material prior to testing: prepared daily in the vehicle
- Final dilution of a dissolved solid, stock liquid or gel: 0.25, 0.50, 1.0 and 2.0 % (w/v)
- Final preparation of a solid: dissolved in vehicle

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CBA/Ca mouse were obtained from Harlan, UK
- Age at study initiation: 8 weeks
- Weight at study initiation: 18 – 23 g on the arrival of animals.
- Housing: Animals were housed in groups of 5 of the same dose group in plastic cages (265 x160 x 140 mm). Bedding comprised 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 libidum (Diet reference A04-C10). Diet was sterilized by irradiation and analyzed for chemical and bacterial contaminants.
- Water: Filtered (0.2µm) mains drinking water ad libidum (via bottle). Water was analyzed at least once a year for chemical contaminants and at least twice a year for bacterial contaminants.
- Contaminants: 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: Animals were acclimatized for 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: 22 ± 2°C
- Relative humidity: 55±15%
- Air changes: 12 air changes per hour
- Photoperiod: 12 hours light (artificial)/12 hours dark
IN-LIFE DATES: From: March 19, 1999 To: March 24, 1999

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Remarks:

DMSO

Concentration:

0 (DMSO), 0.25%, 0.50%, 1% and 2% (w/v)

No. of animals per dose:

5 female animals/dose group

Details on study design:

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The criterion for a positive response was that the test substance or positive control elicits a 3-fold or greater increase in isotope incorporation relative to the negative control group (stimulation index of ≥3).

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment Preparation: The test substance and positive control solution were prepared daily as a solution in the DMSO (vehicle) at concentrations of 0.25, 0.50, 1 and 2% (w/v). The test preparations were used within 4 hours of preparation.
- Administration of the test preparations: On Day 1, 2 and 3, 25 µL of the test substance preparation at concentrations of 0, 0.25, 0.50, 1 and 2% (w/v) or vehicle was applied on the dorsal surface of both ears. A hair dryer was used for about 5 minute to dry mice ears.
The assay of the positive control (P-phenylenediamene, PPD) was performed with 5 animals per sub-group in the study number 762/018. The results were also included in this report in order to compare the effects obtained with the test substance.

OBSERVATIONS
- Morbidity/mortality: All animals were observed at least once daily.
- Clinical signs: Animals were observed daily. During the treatment period, animals were observed before and at least once after dosing to detect any clinical signs or reaction to treatment.

EVALUATION OF CELL PROLIFERATION:
On Day 6, 250 µL of phosphate buffered saline (PBS) containing 20 µCi of [3H] methyl thymidine was injected intravenously. Five hour later, the mice were sacrificed by carbon dioxide inhalation and the draining auricular lymph node excised. A single cell suspension of lymph node cells was prepared for each sub group. Cells were washed twice with PBS and precipitated with ice cold 5% trichloro-acetic acid (TCA). Approximately 18 hours later, the pellets were resuspended in 1 mL of TCA and transferred to 10 mL scintillation cocktail. Incorporation of 3H-methyl thymidine was measured by liquid scintillation technique using a TRI-CARB 2700TR Packard liquid scintillation analyser.

Positive control substance(s):

other: p-phenylenediamine(PPD)

Statistics:

As the lymph nodes were pooled for each group, no statistical analysis was performed (one value per group only).

Results and discussion

Positive control results:

- Stimulation indices were 5.47, 12.39, 19.12 and 7.07 at 0.25%, 0.50%, 1% and 2% (w/v), respectively.
- DPM: 20568.5, 46652.5, 71978.4 and 26597.7 at 0.25%, 0.50%, 1% and 2% (w/v), respectively.
- Corrected DPM: 7.27, 16.48, 25.43 and 9.40 at 0.25%, 0.50%, 1% and 2% (w/v), respectively

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: - Stimulation indices were 1.29, 1.03, 1.12 and 1.42 at 0.25%, 0.50%, 1% and 2% (w/v), respectively. These stimulations indices were not greater than 3, hence the response was considered as negative.

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: - DPM: 17373.5, 13898.6, 15087.3 and 19073.2 at 0.25%, 0.50%, 1% and 2% (w/v), respectively. - Corrected DPM: 5.61, 4.49, 4.87 and 6.16 at 0.25%, 0.50%, 1% and 2% (w/v), respectively.

Results 3

Key result

Parameter:

SI

Value:

1.29

Test group / Remarks:

Test item 0.25%

Results 4

Key result

Parameter:

SI

Value:

1.03

Test group / Remarks:

Test item 0.50%

Results 5

Key result

Parameter:

SI

Value:

1.12

Test group / Remarks:

Test item at 1%

Results 6

Key result

Parameter:

SI

Value:

1.42

Test group / Remarks:

Test item at 2%

Any other information on results incl. tables

Evaluation of the cell proliferation

Evaluation of the cell proliferation
Dose		DPM	Corrected DPM (x10E-4)	Stimulation Index
Negative control		13448.2	4.34
Test item	0.25	17373.5	5.61	1.29
	0.5	13898.6	4.49	1.03
	1	15087.3	4.87	1.12
	2.0	19073.2	6.16	1.42
Positive control	0.25	20568.5	7.27	5.47
	0.5	46652.5	16.48	12.39
	1	71978.4	25.43	19.12
	2.0	26597.7	9.40	7.07

 

Mortality:No mortality was observed during the course of the study.

Clinical Signs:No treatment-related clinical signs were observed.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

Migrated information Criteria used for interpretation of results: expert judgment

Conclusions:

Lehmann Blau (1-methoxy-2-amino-4-β-hydroxyethylaminobenzene-dihydrochloride) did not induced skin sensitization in the Local Lymph Node Assay (LLNA) when tested up to 2% (w/v) solution in DMSO. However, the dose level used were too low for substance sensitization and hypersensitivity evaluation.

Executive summary:

The study was performed to assess the skin sensitization potential of Lehmann Blau (1-methoxy-2-amino-4-β-hydroxyethylaminobenzene-dihydrochloride) by following method comparable to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay). The study was conducted in general compliance with the methodology published in Methods in Immunotoxicology, Vol. 2, pages 279-290, 1975

25 μl of negative control (DMSO, the vehicle), 0.25, 0.5, 1 and 2% of Lehmann-Blau in DMSO w/v was applied to the surface of the ear of five female CBA/Ca mice per group for three consecutive days. After application, the ears were dried with a hair dryer for five minutes. As the positive control, p-phenylenediamine (PPD) in DMSO at the same dilutions was used in parallel. On day 5, the mice received an intravenous injection of 250 μl phosphate buffered saline containing 25 μCi of [H3] methyl thymidine. Approximately 5 hours later, the mice were killed by CO2 inhalation and the draining auricular lymph nodes removed and weighed. After preparing a single cell suspension for each mouse, cells were precipitated by TCA and the radioactivity determined by means of liquid scintillation counting as disintegrations per minutes (dpm). The mean dpm per treated group was determined and the stimulation index (SI) – the test item compared to the concurrent vehicle control – calculated.

No concentration dependent increase in the mean SI values (1.29, 1.03, 1.12, 1.42) could be detected for the 4 consecutive concentrations of Lehmann-Blau in DMSO. The sensitivity of the test system was shown by the positive control, PPD, for which the SI were 5.47, 12.39, 19.12 and 7.07 respectively for the 4 consecutive concentrations.

There was no indication of skin sensitisation by Lehmann-Blau at up to 2% in DMSO. An EC3 value could not be calculated. Indeed, LLNA indicates that the substance is not allergenic at a concentration of 2% in DMSO.

However, the study was not performed correctly since the induction concentrations used were too low. These doses are not suitable for a robust hazard assessment for classification and labelling.