3-hydroxy-2'-methoxy-2-naphthanilide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31.08.-06.09.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

3-hydroxy-2'-methoxy-2-naphthanilide

EC Number:

205-206-6

EC Name:

3-hydroxy-2'-methoxy-2-naphthanilide

Cas Number:

135-62-6

Molecular formula:

C18H14NNaO3

IUPAC Name:

3-hydroxy-2'-methoxy-2-naphthanilide

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
-strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 6-7 weeks (beginning of acclimatization)
- Weight at study initiation: 17.2 g to 21.8 g
- Housing: single caging, Makrolon Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 84 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0.1, 0.2, 0.4, 1 and 5% (w/v)

No. of animals per dose:

4 animals per treatment group, 4 animals in the control group

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each
ear lobe with test item concentrations of 0.1, 0.2, 0.4, 1 and 5% (w/v) in DMSO. The application
volume, 25 μL/ear/day, was spread over the entire dorsal surface (diameter approx. 8 mm) of each
ear lobe once daily for three consecutive days. A further group of mice (control animals) was treated
with an equivalent volume of the relevant vehicle alone.

Administration of 3H-methyl-thymidine:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing
20.4 μCi of 3H-methyl thymidine (equivalent to 81.5 μCi/mL 3HTdR) were injected into each test and
control mouse via the tail vein.

Determination of incorporated 3HTdR:
Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal
injection of Na-thiopental.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal).
After preparation of the cells, precipitation and resuspension, the level of 3HTdR incorporation was measured in a
β-scintillation counter. The β-scintillation counter expresses 3HTdR incorporation as the number of
radioactive disintegrations per minute.

Interpretation of data:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or
greater than that recorded in control mice, as indicated by the Stimulation Index.
- data are compatible with a conventional dose response, although allowance must be made
(especially at high topical concentrations) for either local toxicity or immunological suppression.

Observations:
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability once daily (week day) from experimental start to necropsy.
Body weights prior to the first application and prior to treatment with 3HTdR.
Clinical signs (local / systemic) at 1 – 2 hours after each application. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated.

A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the coordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

Results and discussion

Positive control results:

EC3 = 18.2 % (w/v)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.93

Test group / Remarks:

0.1% (w/v)

Results 2

Key result

Parameter:

SI

Value:

2.71

Test group / Remarks:

0.2% (w/v)

Results 3

Key result

Parameter:

SI

Value:

4.62

Test group / Remarks:

0.4% (w/v)

Remarks on result:

other: skin sensitising

Results 4

Key result

Parameter:

SI

Value:

4.28

Test group / Remarks:

1% (w/v)

Remarks on result:

other: skin sensitising

Results 5

Key result

Parameter:

SI

Value:

8

Test group / Remarks:

5% (w/v)

Remarks on result:

other: skin sensitising

Results 6

Key result

Parameter:

EC3

Value:

0.2

Remarks on result:

other: skin sensitising

Any other information on results incl. tables

No deaths, clinical signs or abnormal body weight development were observed during the study period.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was found to be a skin sensitiser in the LLNA when tested at 0, 0.1, 0.2, 0.4, 1.0 and 5.0% in DMSO (w/v).
An EC3 value is calculated to be 0.2%

Executive summary:

In order to study a possible contact allergenic potential of Naphtol AS-OL, five groups each of four female mice were treated daily with the test item at concentrations of 0.1, 0.2, 0.4, 1 and 5% (w/v) in DMSO by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (DMSO) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 1.93, 2.71, 4.62, 4.28 and 8.00 were determined with the test item at concentrations of 0.1, 0.2, 0.4, 1 and 5% (w/v) in DMSO. The EC3 value calculated was 0.2 % (w/v).

The test item Naphtol AS-OL was found to be a skin sensitizer under the described conditions.

The study was performed in compliance to OECD TG 429 and GLP.