5'-chloro-3-hydroxy-2',4'-dimethoxy-2-naphthanilide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.-19.02.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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:

5'-chloro-3-hydroxy-2',4'-dimethoxy-2-naphthanilide

EC Number:

202-182-9

EC Name:

5'-chloro-3-hydroxy-2',4'-dimethoxy-2-naphthanilide

Cas Number:

92-72-8

Molecular formula:

C19H16ClNO4

IUPAC Name:

5'-chloro-3-hydroxy-2',4'-dimethoxy-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 Laboratories B.V., The Netherlands
- Age at study initiation: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 18.3 g to 21.5 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: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light, 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 0.03, 0.3, 1.0, 3.0, 10.0 % (w/v)

No. of animals per dose:

5 animals per treatment group, 5 animals in the control group

Details on study design:

Application of the test item:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear lobe with test item concentrations of 0.03, 0.3, 1, 3, and 10% (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 20.3 μCi of 3H-methyl thymidine (equivalent to 81.2 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 Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal).
After preparation of the cells and precipitation, 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.

Positive control substance(s):

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

Results and discussion

Positive control results:

EC3: 15.7% (w/v)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.06

Test group / Remarks:

test item concentration of 0.03%

Results 2

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

test item concentration of 0.3%

Results 3

Key result

Parameter:

SI

Value:

1.64

Test group / Remarks:

test item concentration of 1%

Results 4

Key result

Parameter:

SI

Value:

3.02

Test group / Remarks:

test item concentration 3%

Remarks on result:

other: skin sensitiser

Results 5

Key result

Parameter:

SI

Value:

3.25

Test group / Remarks:

test item concentration 10%

Remarks on result:

other: skin sensitiser

Results 6

Key result

Parameter:

EC3

Value:

3

Remarks on result:

other: skin sensitiser

Any other information on results incl. tables

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Conclusions:

The test item was found to be a skin sensitiser in the LLNA when tested at 0.03, 0.3, 1.0, 3.0 and 10.0% in DMSO (w/v).
An EC3 value is calculated to be 3.0% (w/v).

Executive summary:

In order to study a possible contact allergenic potential of Naphtol AS-ITR, five groups each of five female mice were treated daily with the test item at concentrations of 0.03, 0.3, 1, 3, and 10% (w/v) in dimethylsulphoxide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of five mice was treated with the vehicle (dimethylsulphoxide) 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 animal. 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.

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

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 1.06, 1.30, 1.64, 3.02 and 3.25 were determined with the test item at concentrations of 0.03, 0.3, 1, 3, and 10% in dimethylsulphoxide. The EC3 value calculated was 3.0%.

Statistical evaluation was performed by Dunnett-Test (ANOVA) (p > 0.05). At the concentrations of 3 and 10 % of test item a statistically significant difference of DPM per animal was determined (p = <0.001).

The test item Naphtol AS-ITR was found to be a skin sensitiser under the described conditions.
The study was performed in compliance to OECD TG 429 and GLP.