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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13.-20.02.2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD guideline not specified in the report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

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: 8-9 weeks (beginning of treatment)
- Weight at study initiation: 18.3 g to 23.7 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs 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:
Topical Application:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface
of each ear lobe (left and right) with different test item concentrations of 0.03, 0.3, 1, 3, and
10% (w/v) in dimethylsulfoxide. The application volume, 25 μl, 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 was treated with an equivalent volume of the relevant vehicle alone (control
animals).

Administration of 3H-Methyl Thymidine:
3H-methyl thymidine (3HTdR); specific activity 2 Ci/mmol; concentration 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 μl of
81.2 μCi/ml 3HTdR (corresponds to 16.2 μCi 3HTdR per mouse) by intravenous injection
via a 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, 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 Raw Data:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to 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.
- Second, that the 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) once daily (week day). 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.

The ANOVA (Dunnett-test) was conducted to assess whether the difference is statistically
significant between test item groups and negative control (vehicle) group. Statistical
significance was at the five per cent level (p < 0.05). However, both biological and
statistical significance were considered together.

Results and discussion

Positive control results:
EC3: 15.7% (w/v) (experiment performed in February 2008)

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.13
Test group / Remarks:
0.03%
Key result
Parameter:
SI
Value:
1.95
Test group / Remarks:
0.3%
Key result
Parameter:
SI
Value:
1.68
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
1.75
Test group / Remarks:
3%
Key result
Parameter:
SI
Value:
2.28
Test group / Remarks:
10%
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
all S.I.´s are below 3

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:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item was found not 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 could not be calculated, since all S.I.´s are below 3.
Executive summary:

In order to study a possible contact allergenic potential of Naphtol AS-RL, five groups each

of five female mice were treated daily with the test item at a concentrations of 0.03, 0.3, 1,

3, and 10% (w/v) in dimethylsulfoxide 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 (dimethylsulfoxide) 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 1.13, 1.95, 1.68, 1.75 and 2.28 were determined with

the test item at concentrations of 0.03, 0.3, 1, 3, and 10% in dimethylsulfoxide.

The EC3 value could not be calculated, since none of the tested concentrations induced

an S.I. greater than 3.

Statistical evaluation was performed by Dunnett-Test (ANOVA) (p > 0.05). At the

concentrations of 0.3, 1, 3, and 10 % of test item a statistically significant difference of

DPM per animal was determined (p = <0.001). Nevertheless, the threshold S.I. value of 3

was not reached with any of the tested concentrations.

The test item Naphtol AS-RL was found to be not a skin sensitiser under the described

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