Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 2009 to 11 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19 August 2008; Date of signature: 04 March 2009
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : H-CB sodium salt
Description : blue solid
Lot number : MB-2
Date received : 04 December 2008
Storage conditions : room temperature in the dark

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca (CBA/CaOlaHsd)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories UK Limited, Bicester, axon, 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: Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study
- Water: Free access to mains tap water was allowed throughout the study
- Acclimation period: At least five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): Approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

IN-LIFE DATES: FromDay 0 : To: Termination

Study design: in vivo (LLNA)

Vehicle:
other: Prepared as a solution in 1% pluronic L92 in distilled water. The vehicle Determination Record can be found in Table 1. in the information on materials and methods section below.
Concentration:
Each group was exposed to concentrations of 25%, 10% or 5% w/w (in 1% pluronic L92 in distilled water)
No. of animals per dose:
Groups of five mice were treated
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 1% pluronic L92 in distilled water
- Irritation: The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Any signs of toxicity or excessive local irritation noted during this period were recorded.
- Lymph node proliferation response: Not performed for preliminary screening test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- 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 nodes from each individual animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to
that recorded for the control nodes (Stimulation Index).

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:
Groups of five mice were treated with the test material at concentrations of 25%, 10% or 5% w/w in 1% pluronic L92 in distilled water. The preliminary screening test suggested that the test material would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. The mice were treated by daily application of 25 µI of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). 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.

A further group of five mice received the vehicle alone in the same manner.

Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µI of phosphate buffered
saline (PBS) containing 3H-methyl thymidine eHTdR:80µCi/ml, specific activity 2.0 Ci/mmol,GE Healthcare UK Ltd) giving a total of 20 µCi to each
mouse.
Positive control substance(s):
other: 2,4-Dinitrobenzenesulfonic acid, sodium salt
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships by analysis of homogeneity of variance followed by one way analysis of variance (ANOVA). In the event of a significant result from the ANOVA, pairwise comparisons were performed between control and treated groups. For homogenous datasets Dunnett's Multiple Comparison test was used and for non-homogenous datasets Dunnett's T3 Multiple Comparison
Method was used.

Probability values (p) are presented as follows:
P<0.001 = ***
P<0.01 = **
P<0.05 = *
P≥0.05 = (not significant)


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
10 13.71 Positive

Conclusion. 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 test material at concentrations of 5%, 10% and 25% w/w in 1% pluronic L92 in distilled water. The stimulation index (SI) results are given in Table 2.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: The radioactive disintegrations per minute (dpm) per lymph node and the stimulation index (SI) are given in Table 2 in Attachment 1 found in the attachments section below.

Any other information on results incl. tables

Clinical Observations and Mortality Data Individual clinical observations and mortality data for test and control animals are given in Table 3 of attachment 1 found in the attachments section below. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test. Blue coloured staining on the ears and fur was noted, post dose on Days 1 to 3, in animals treated with the test material at a concentration of 25% w/w in 1% pluronic L92 in distilled water. Bodyweight 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.

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:

Introduction.

A 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 Regulation (EC) No. 440/2008

• United States Environmental Protection Agency Health Effects Test Guidelines OPPTS 870.2600 Skin Sensitisation March 2003

Methods. Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µI (25 µI per ear) of the test material as a solution in 1% pluronic L92 in distilled water at concentrations of 25%, 10% or 5% w/w. A further group of five animals was treated with 1% pluronic L92 in distilled water alone.

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

5

1.12

Negative

10

0.98

Negative

25

1.16

Negative

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