5-[(2-hydroxyethyl)amino]-o-cresol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15 December 2003 to 25 November 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

Guideline:

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

Version / remarks:

24th April 2002

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

5-[(2-hydroxyethyl)amino]-o-cresol

EC Number:

259-583-7

EC Name:

5-[(2-hydroxyethyl)amino]-o-cresol

Cas Number:

55302-96-0

Molecular formula:

C9H13NO2

IUPAC Name:

5-[(2-hydroxyethyl)amino]-2-methylphenol

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Analytical purity: 99.3% (HPLC; Area % without response factor, UV detection)
- Purity test date: August 2004
- Lot/batch No.: 0121442
- Expiration date of the lot/batch: September 2005
- Titre: 99.8% (based upon alkalinity in g/100g)
- Description: beige powder
- Trade name: IMEXINE OAG
- Labeling code: 2948

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +4°C, protected from light and under nitrogen gas
- Stability under test conditions: The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within the 4 hours following the preparation according to the known stability results.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: no data
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation (mean ± standard deviation): 21.1 ± 1.3 g
- Housing: animals were housed individually in disposable crystal polystyrene cages (22.00 cm x 8.50 cm x 8.00 cm). Each cage contained autoclaved sawdust (SICSA, Alfortville, France). Sawdust was analysed by the supplier for composition and contaminant levels.
- Diet: ad libitum to A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France). Each batch of diet was analyzed for composition and contaminant level by the supplier (diet formula is provided in the study report).
- Water: ad libitum to tap water (filtered using a 0.22 micron filter) contained in bottles. Bacteriological and chemical analyses of water, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines) awere performed regularly by external laboratories.
No contaminants are known to be present in the diet, drinking water or sawdust at levels which may be expected to interfere with or prejudice the outcome of the study.
- Acclimation period: at least 5 days before the beginning of the study
- Indication of any skin lesions: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr): 12 /12
- Photoperiod (hrs dark / hrs light): 12 cycles of filtered, non-recycled air

The temperature and relative humidity are under continuous control and recording. The records are checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

2.5, 5, 10, 25 and 50% w/v in dimethylformamide

No. of animals per dose:

4 females for the preliminary test, 28 females for the main test (4 females per concentration group)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was prepared as a solution in the vehicle at the chosen concentrations. The test item dosage forms were prepared extemporaneously under nitrogen atmosphere and were stored protected from light and under nitrogen gas before use. They were used within the 4 hours following the preparation according to the known stability results.
- To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was performed on a small number of animals, as follows:
• the test item was prepared at the concentrations of 50, 25, 10 and 5%,
• for 3 consecutive days, the animals received applications of 25 μL of the dosage form preparations to the external surface of both ears (one concentration per ear),
• measurement of the ear thickness (using a micrometer) was performed each day before treatment and 24 hours after the last application.

MAIN STUDY
- The concentrations of test item were selected according to the criteria specified in the International Guideline and on the basis of the results of the solubility and preliminary assays and were as follows:

• 4 females treated with the vehicle (dimethylformamide)
• 4 females treated with the test item at 2.5%
• 4 females treated with the test item at 5%
• 4 females treated with the test item at 10%
• 4 females treated with the test item at 25%
• 4 females treated with the test item at 50%
• 4 females treated with the postive control HCA (α-hexylcinnamaldehyde) at 25%

TREATMENT PREPARATION AND ADMINISTRATION:
- On days 1, 2 and 3, a dose-volume of 25 μL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
- In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
- No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.

CLINICAL EXAMINATIONS:
- Animals were observed at least once a day during the study for clinical signs, signs of morbidity or mortality.
- Animals were weighed individually on the first day of the study (day 1) and on the day of sacrifice (day 6).
- Ear thickness measurements and recording of local reactions: performed in order to assess any possible irritant effect of the test item, as possible irritancy may be involved in false positive lymphoproliferative responses. On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of groups 1 to 6 was measured using a micrometer. No measurement of ear thickness was performed for the animals of the positive control group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item, …) was noted. The irritation level of the test item was determined according to table 1 in "Any other information on materials and methods inl. tables".

PROLIFERATION ASSAY:
- Intravenous injection of 3H-TdR and sampling of auricular lymph nodes: Lymph node cell proliferative responses were measured as described by Kimber and Dearman (1991). On day 6, all animals of all groups received a single intravenous injection of 250 μL of 0.9% NaCl containing 20 μCi of 3H-TdR (specific activity of 25 Ci/mmol) via the tail vein. Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
- Preparation of auricular lymph node cell suspensions and determination of proliferation: For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting. The results were expressed as disintegration's/mn (dpm) per group and per node. Stimulation Indices (SI) were calculated according to the following formula: SI= dpm of treated group / dpm of control group
- Interpretation of results: The test item was considered as a skin sensitizer when the SI for a dose group is ≥ 3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness were also taken into account for the interpretation of results.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

A moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 8.39) were noted. The study was therefore considered valid.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.81

Test group / Remarks:

Test item at 2.5%

Results 2

Key result

Parameter:

SI

Value:

1.31

Test group / Remarks:

Test item at 5%

Results 3

Key result

Parameter:

SI

Value:

0.61

Test group / Remarks:

Test item at 10%

Results 4

Key result

Parameter:

SI

Value:

0.53

Test group / Remarks:

Test item at 25%

Results 5

Key result

Parameter:

SI

Value:

0.44

Test group / Remarks:

Test item at 50%

Results 6

Key result

Parameter:

SI

Value:

8.39

Test group / Remarks:

Positive control (HCA)

Cellular proliferation data / Observations:

CHOICE OF THE VEHICLE
- The choice of the vehicle was based on the results of a solubility assay. The vehicle used in the study was dimethylfomamide (DMF): a homogeneous solution was obtained at the maximum concentration of 50%

PRELIMINARY TEST
-Since the test item was non-irritant in the preliminary test, the highest concentration retained for the main test was the maximal practicable concentration (i.e. 50%), according to the criteria specified in the International Guideline.

CELLULAR PROLIFERATION DATA
- Results of proliferation assay are presented in table 2 (in "Any other information on results incl.tables".
- The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of 3H-TdR. The cell viability was higher than 80% in each group.
- No positive lymphoproliferative responses and no dose-response relationship were observed in the test item treated groups.

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS:
- No clinical signs and no mortality were observed during the study.
- No cutaneous reactions and no noteworthy increases in ear thickness were observed at any of the tested concentrations.

BODY WEIGHTS
- The body weight change of the treated animals was similar to that of the control animals

Any other information on results incl. tables

Table 2. Study results

Treatment and concentrations	Cell count		Viability		Amount of cells (x 10E6 cells)	Cellularity index	Number of nodes per group	dpm per group	dpm per node	Stimulation index (SI)	Increase in ear thickness (% between day 1 and day 6)	EC3 value	Irritation classe
	viable	dead
Dimethylformamide 0	125	8	93.98		6.25		8	523.14	65.39		4.17
A031 2.5%	181	10	94.76		9.05	1.45	8	424.08	53.01	0.81	9.38	NA	I
A031 5%	164	9	94.80		8.20	1.31	8	683.96	85.50	1.31	3.00
A031 10%	99	13	88.39		4.95	0.79	8	318.63	39.83	0.61	6.12
A031 25%	108	6	94.74		5.40	0.86	8	275.87	34.48	0.53	4.08
A031 50%	81	11	88.04		4.05	0.65	8	231.24	28.91	0.44	4.17
HCA 25%	300	61	83.10		30.00	4.80	8	4391.38	548.92	8.39

dpm = disintegrations per minute

Viability = viable cells / ( viable cells + dead cells)

Cellularity index = amount of cells (x106 cells) in the treated group / amount of cells (x106 cells) in the vehicle group

Stimulation index = dpm of treated group / dpm of control group

EC3 value = theorical concentration resulting in a SI value of 3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study , the test item 2-methyl-5-hydroxyethylamino-phenol did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay. Thus, the test item is not classified as a skin sensitizer according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.

Executive summary:

This GLP-compliant study was performed to assess the potential of 2-Methyl-5-hydroxyethylaminophenol to induce delayed contact hypersensitivity, according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) (dated 24th April 2002) guideline.

Material and methods

A preliminary irritation test was first performed in order to define the concentrations of test item to be used in the main test. Dimethylformamide was selected as a vehicle in a solubility study showing that the test item was non-soluble in other recommended vehicles, and that 50% (w/v) test material in dimethylformamide was the maximal practicable concentration. This concentration was non-irritant in the preliminary test.

Twenty-eight female mice were used in the main study allocated to seven groups of 4 animals each: 5 treated groups receiving the test item at the concentrations of 2.5, 5, 10, 25 and 50% (w/v) in the vehicle (dimethylformamide); a negative control group receiving dimethylformamide alone, and a positive control group receiving alpha-hexylcinnamaldehyde (a moderate sensitizer) at 25 % (v/v) in dimethylformamide.

The test substance or the positive control were applied over the ears (25 μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph nodes draining the application sites was measured by incorporation of tritiated methyl thymidine on day 6. The values obtained were used to calculate stimulation indices (SI). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

Results

No cutaneous reactions and increases in ear thickness were observed in the animals of the treated groups. No lymphoproliferative responses were observed in the test substance groups, while lymphoproliferation was observed with alpha-hexylcinnamaldehyde at 25% (SI value of 8.39).

Conclusion

Under the experimental conditions of this study, the test item 2-methyl-5-hydroxyethylamino-phenol did not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay. Thus, the test item is not classified as a skin sensitizer according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) criteria.