Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10/12/2008 to 16/12/2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
No further information required.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, Oxon, UK.
- Age at study initiation: Eight to twelve weeks old
- Weight at study initiation: 15 to 23 g
- Housing: The animals were individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet (e.g. ad libitum): Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: Minimum of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): The rate of air exchange was approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Challenge controls:
n/a
Positive control substance(s):
yes
Remarks:
2,4-Dinitrobenzenesulfonic acid, sodium salt

Study design: in vivo (LLNA)

Vehicle:
other: 1% pluronic L92 in distilled water.
Concentration:
1% pluronic L92 in distilled water.
No. of animals per dose:
4 mice
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: For the purpose of the study, the test material was freshly prepared as a solution in 1% pluronic L92 in distilled water. This vehicle was chosen as it produced the highest concentration that was suitable for dosing.
- Irritation: Information available suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration.
- Lymph node proliferation response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002) and Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/731EC.

- Criteria used to consider a positive response: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node and as the ratio of ^3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes. The test material will be regarded as a sensitiser if at least one concentration of the test material results in a threefold or greater increase in ^3HTdR incorporation compared to control values. Any test material failing to produce a threefold or greater increase in ^3HTdR incorporation will be classified as a "non-sensitiser".

TREATMENT PREPARATION AND ADMINISTRATION:
MAIN STUDY
- No. of exposures: 3
- Test groups: Groups of four mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water.
- Control group: A further group of four mice received the vehicle alone in the same manner.
- Site: The test material formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
- Frequency of applications: The mice were treated by daily application of 25 µl of the appropriate concentration of the test material to the dorsal surface of each ear.
- Duration: three consecutive days (Days 1, 2, 3).
- Concentrations: 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water.
Positive control substance(s):
other: 2,4-Dinitrobenzenesulfonic acid, sodium salt
Statistics:
n/a

Results and discussion

Positive control results:
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:
Concentration (% w/w) in 1% pluronic L92 in distilled water Stimulation Index Result
1 0.96 Negative
5 4.29 Positive
10 13.15 Positive

Therefore, 2,4-Dinitrobenzenesulfonic acid, sodium salt was considered to be a sensitiser under the conditions of the test.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: A stimulation index of less than 3 was recorded for the three concentrations of the test material (25%, 10% and 5% w/w in 1% pluronic L92 in distilled water).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (%w/w) in 1% pluronic L92 in distilled water dpm dpm/Node(a) Stimulation Index(b) Result Vehicle 5383.57 672.95 na na 5 5151.07 643.88 0.96 Negative 10 2869.31 358.66 0.53 Negative 25 5486.88 685.86 1.02 Negative dpm = Disintegrations per minute a = Disintegrations per minute/node obtained by dividing the disintegrations per minute value by 8 (total number of lymph nodes) b = Stimulation Index of 3.0 or greater indicates a positive result na = Not applicable

Any other information on results incl. tables

A stimulation index of less than 3 was recorded for the three concentrations of the test material (25%, 10% and 5% w/w in 1% pluronic L92 in distilled water).

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Blue coloured staining of the ears and fur was noted, post dose on Days 1 to 3, in animals treated with the test material at concentrations of 25% or 10% w/w in 1% pluronic L92 in distilled water.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
Executive summary:

In a local lymph node assay in the mouse study (Harlan project number: 0959/0230) the test material was considered to be a non-sensitiser under the conditions of the test.

The study was performed to assess the skin sensitisation potential of the test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following:

 - OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 24 April 2002).

 -  Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Directive 2004/731EC.

A stimulation index of less than 3 was recorded for the three concentrations of the test material (25%, 10% and 5% w/w in 1% pluronic L92 in distilled water).

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Blue coloured staining of the ears and fur was noted, post dose on Days 1 to 3, in animals treated with the test material at concentrations of 25% or 10% w/w in 1% pluronic L92 in distilled water.

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

The test material was considered to be a non-sensitiser under the conditions of the test.