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Description of key information

A key study completed under GLP conditions and in accordance with OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2005 to 05 May 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other:
Sex:
female
Details on test animals and environmental conditions:
At the start of the study the mice were in the weight range of 15 to 23 grams and were eight to twelve weeks old. One animal was above the maximum weight per standard test method (23 grams) but this was not considered to affect the integrity of the study. Free access to mains drinking water and food was allowed through out the study. The temperature and relative humidity were set to achieve limits of 19 to 25 C and 30 to 70% respectively. The rate of air exchange was at least 15 changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours of darkness.
Vehicle:
dimethylformamide
Concentration:
test material at concentrations of 25%, 10% or 5% w/w
No. of animals per dose:
Three groups of five animals each were treated at each concentration group and a further group of five animals were treated as a control group with dimethyl formamide alone.
Details on study design:
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 μl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) by using a micropipette whose tip is used to spread the formulation over the dorsal surface of each ear. On Day 6 all mice were injected with 250 ul of a phophate buffered saline solution containing 3H-methyl thymidine (3HTdR ) to the tail vein giving a total of 20 uCi to each mouse. All animals were observed twice daily on Day 1, 2 and 3 and once daily on days 4 ,5, and 6. Any signs of toxicity or ill health were noted. Body weights of each mouse were recorded before dosing and before termination. Five hours after administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and their auricular lymph nodes were excised and a 1 ml of phosphate buffered saline was added to each set of lymph nodes. Preparation of a single cell suspension was completed for the lymph nodes cells for each individual animal following standard procedures. Determination of the 3HTdR incorporation was completed by measuring radioactive disintegration for each animal's lymph node cells by using a beta-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Group mean values for disintegration per minute (dpm) and standard deviations where appropriate are completed. Individual and dose mean dpm values were assessed for dose response relationships by analysis of homogenicity of variance followed by one way analysis of variance (ANOVA). If significant results were determined from the ANOVA then 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.
Positive control results:
α-Hexylcinnamaldehyde, Tech, 85% was considered to be a sensitiser under the conditions of the test with SI rates of 5%- 2.76( Negative), at 10% SI- 3.34 (Positive) and 25% a SI of 8.91% (Positive).
Parameter:
other: disintegrations per minute (DPM)
Value:
> 1 135.46 - < 1 821.76
Test group / Remarks:
all test groups (5%, 10% and 25%)
Remarks on result:
other: see Remark
Remarks:
Concentration of test material in the vehicle at 5 % resulted in a mean disintegration of 1275.64 dpm which is a negative result.Concentration of test material in the vehicle at 10 % resulted in a mean disintegration of 1135.46 dpm which is a negative result. Concentration of test material in the vehicle at 25 % resulted in a mean disintegration of 1821.76 dpm which is a negative result. The results of the statistical analysis of the data indicated there was no significant difference between the control group and the test groups.
Parameter:
SI
Value:
1.28
Test group / Remarks:
5%
Remarks on result:
other: negative
Parameter:
SI
Value:
1.14
Test group / Remarks:
10%
Remarks on result:
other: negative
Parameter:
SI
Value:
1.83
Test group / Remarks:
25%
Remarks on result:
other: negative

There were no deaths. No signs of systemic toxicity were noted in the test or control animals

during the test. Residual test material on the ears was noted on Days 1 to 3, one hour post dosing,

in all animals treated with the test material at concentrations of 10% and 25% w/w.

 

Concentration (% w/w) in

dimethyl formamide

 Stimulation index (SI)

 Result

 5

 1.28

 Negative

 10

 1.14

 Negative

 25

 1.83

 Negative

Interpretation of results:
not sensitising
Conclusions:
The test material was considered to be a non-sensitiser under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Concentration of test material in the vehicle at 5 % resulted in a stimulation index (SI) of 1.28 which is a negative result. Concentration of test material in the vehicle at 10 % resulted in a stimulation index (SI) of 1.14 which is a negative result. Concentration of test material in the vehicle at 25 % resulted in a stimulation index (SI) of 1.83 which is a negative result. A stimulation index of less than 3 was recorded for the three concentrations of the test material and the substance is considered not a skin sensitizer according to EU CLP regulations.