Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 September 2009 – 16 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to EC and OECD test guidelines, and in compliance with GLP.

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)
Remarks:
The study report included a current certificate of GLP compliance for the test facility, issued by the MHRA.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Off-white solid
- Storage condition of test material: aprox. -20 degrees in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd
- Age at study initiation: Approximately 8 to 12 weeks of age prior to dosing on Day 1.
- Weight at study initiation: 18.2 to 21.5 g.
- Housing:The animals were housed in pairs in polycarbonate cages with woodflake bedding. The mice were also given Nestlets for environmental
enrichment.
- Diet (e.g. ad libitum): The animals were allowed free access to a standard pelleted rodent diet (Rat & Mouse No. 1 Maintenance diet).
- Water (e.g. ad libitum):Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light


IN-LIFE DATES: From: 03 September 2009 (date of animal allocation to groups) To: 14 September 2009

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0 (Dimethylformamide control), 10, 25, and 50% w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Very thick suspensions were formed at 50% (w/v) in acetone: olive oil (4:1 (v/v)), ethyl methyl ketone and propylene glycol,
which were too viscous for dose administration. Dimethyl sulphoxide at 50% (w/v) and acetone:olive oil (4:1 (v/v)) and ethyl methyl ketone at 25%
formed bitty suspensions which passed the needle test. Dimethylformamide formed a pale orange/brown suspension at 50% (w/v) which was
satisfactory for dose administration.



MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test substance is regarded as a sensitizer if at least one concentration of the test substance
results in a three-fold greater increase in 3HTdR incorporation compared to control values. (3HTdR = 3H-methyl Thymidine).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
As the low concentration of the test article resulted in a stimulation index of greater than 3, the EC3 value was extrapolated from the two lowest dose utilized. The extrapolated EC3 was calculated by log-linear interpolation between these two points on a plane where the x-axis represents the dose level and the y-axis represents the SI. The point with the higher SI is denoted (a, b) and the point with the lower SI is denoted (c, d). The formula used was as follows:
EC3 = 2(log2(c)+(3-d)/(b-d)x(log2(a)-log2(c)))

Results and discussion

Positive control results:
The test/control ratio for the positive control substance hexyl cinnamic aldehyde (HCA) was 4.2, which demonstrates the reliability and sensitivity of
this assay to detect sensitization potential in this laboratory.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The test/control ratios obtained for 10, 25 and 50% w/v test substance were 3.9, 7.3 and 4.1, respectively.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control (DMF) - 2032.0 (=254.0 dpm/node, 8 nodes) 10% (w/v) - 7932.0 (=991.5 dpm/node, 8 nodes) 25% (w/v) - 14779.0 (=1847.4 dpm/node, 8 nodes) 50% (w/v) - 7332.0 (=1047.4 dpm/node, 7 nodes) HCA (positive control) - 8613.0 (=1076.6 dpm/node, 8 nodes)

Any other information on results incl. tables

As a test/control ratio of 3 or more was recorded for all three concentrations tested, the test substance was considered to have the potential to cause skin sensitization (delayed contact hypersensitivity). The EC3 was calculated and determined to be 3.6% w/v (36 mg/mL).

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
The test substance is regarded as a potential skin sensitizer.
Executive summary:

A localised lymph node assay (LLNA) study was conducted by Huntingdon Life Sciences, UK, to determine the potential for skin sensitization of the test substance. The study was conducted according to OECD 429 and EEC B.42 Test Guidelines, and was performed in compliance with GLP. The test substance is regarded as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values.

The test substance was found to generate a test/control ratio greater than 3 at each concentration tested (10, 25, and 50% (w/v)), and so the test substance was considered to have the potential to cause skin sensitization (delayed contact hypersensitivity).