Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-07-12 to 2004-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 24 April, 2002
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): ad libitum; Certified Rat and Mouse Diet (Code 5LF2) supplied by BCM IPS Limited, London, UK
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
5, 10, 25% (w/w)
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
One female mouse was treated by daily application of 25 µl of the test material at a concentration of 25% (w/w) in dimethyl formamide to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. There were no signs of systemic toxicity or excessive local irritation, only on days 2 and 3 residual test material was noted on the ears one hour post dosing. Based on these results the dose levels selected for the main test were 5%, 10% and 25 % (w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: In a mouse LLNA groups of 5 mice were treated with the test material at concentrations of 5, 10 and 25% (w/w) in dimethyl formamide. 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 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. Five days following the first topical application of the test material (Day 6) all mice were injected via the tail vein with 250 µl of phosphate buffered saline (PBS) containing ³H-methyl thymidine (³HTdR:80µCi/ml, specific activity 2.0 Ci/mmol. Five hours after administration the mice were killed and the lymph node cells were prepared by gentle mechanical disaggregation, several washing steps and resuspension in 5% Trichloroacetic acid in order to precipitate out the radioactive material. After an over night incubation cells were centrifuged and transferred to 10 mL scintillation fluid. The ³HTdR incorporation was measured by β-scintillation
- Criteria used to consider a positive response: 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: For the purpose of the study the test material was freshly prepared in dimethyl formamide. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. 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 for three consecutive days (Days 1, 2, 3).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for dpm and standard deviations where appropriate. Individual and group mean dpm 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.

Results and discussion

Positive control results:
Results of the latest positive control:
5, 10, 25% (v/v) in acetone/olive oil: SI = 1.40, 2.23, 6.09*

*an SI of greater than 3.0 indicates a positive result

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
16.36
Test group / Remarks:
5% test material
Key result
Parameter:
SI
Value:
21.51
Test group / Remarks:
10% test material
Key result
Parameter:
SI
Value:
17.63
Test group / Remarks:
25% test material
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
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 (SI)).

CLINICAL OBSERVATIONS:
There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the study. Residual test material was noted on the ears of the animals treated with the test material at a concentration of 25% w/w in dimethyl formamide one hour post dosing on Days 2 and 3.

BODY WEIGHTS
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.

Any other information on results incl. tables

Table 1: Results of Local Lymph Node Assay (LLNA): Stimulation Index

Sample Description

Test or Control Group

Vehicle

Low

Medium

High

Stimulation Index (SI)

 N/A

 16.36

 21.51

 17.63

EC3b

 

 0.9168

 1.39

 4.254

b)  The Estimated Concentration Three (EC3) is the minimum concentration of a test substance required to elicit a sensitization reaction (produce a stimulation index of three).

SI = Group Mean DPM

EC3 = C + [ (3-D) / (B-D) ] * (A-C)

Where (C,D) are values directly below SI of 3 (C = concentration, D = SI)

and (A,B) are the values above  SI of 3 (A = concentration, B = SI)

Table 2: EC3 value obtained by log-linear extrapolation:

Sample Description

Test or Control Group

Vehicle

Low

Medium

High

Stimulation Index (SI)

 N/A

 16.36

 21.51

 17.63

EC3c

 

 

 0.852

 5.1823E-07

c) Since all measured values exhibited a SI of far above 3 the EC3 value should be obtained by log-linear extrapolation (Ryan et al., 2007) according to the following formula:

EC3ex = 2^[log2 (c) + (3 -d)/(b-d) x (log2 (a) - log2 (c))]

where the point with the value higher than the smallest SI above 3 is denoted (a, b) and the point with the value lower than the smallest SI above 3 is denoted (c, d).

Ryan, Cindy A., et al. "Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency."Cutaneous and ocular toxicology 26(2) (2007): 135-145.

Applicant's summary and conclusion

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
In the present study conducted according to OECD guideline 429 (adopted 24 April 2002), in sum 20 mice of the CBA/Ca strain were dermally exposed to 0, 5, 10 and 25% (w/w) test substance in dimethyl formamide, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). After 5 days following the first topical application the mice were injected with 250 µl PBS containing ³H-methyl-thymidine (2 Ci/mmol). After further 5 h the lymph nodes were dissected and the cells prepared in order to determine the ³HTdR incorporation. Since the Stimulation Index (SI) was above a three-fold increase at every concentration tested and the EC3 obtained by log-linear extrapolation was below 2%, the test item is considered to be a sensitizer based on the obtained results. Thus, the substance needs to be classified as skin sensitizer Category 1A according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).