Registration Dossier

Diss Factsheets

Administrative data

Description of key information

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:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to current guideline under GLP regulations
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus 6174 NL- 5960 AD Horst I The Netherlands
- Age at study initiation: 7 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 14.9 g- 22.0 g (beginning of acclimatization period)
- Housing: Makrolon type-3
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:
other: ethanol:water, 7:3 (v/v))
Concentration:
2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v).
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:

- Irritation: no


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillationbackground DPM was subtracted from test and control raw data.
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
The decision to select a stimulation index of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
ALPHA-HEXYLCINNAMALDEHYDE showed an allergenic potency when tested at
concentration of 25 % (w/v).
EC3 is the estimated concentration for a STIMULATION INDEX of 3. In this study EC3 of 11.3% (w/v) was theoretically calculated with STIMULATION INDICES of 2.6 and 7.1 at test item concentrations of 10 % and 25 % (w/v).
Parameter:
SI
Remarks on result:
other: STIMULATION INDEX. Group 2 test item concentration = 2.5 % (w/v); SI= 1.1 Group 3 test item concentration= 5 % (w/v) ; SI =0.5 Group 4 test item concentration = 10 % (w/v); SI = 1.0 No linear increase of S.l. in relation to the concentrations

VIABILITY / MORTALITY

No deaths occurred during the study period.

CLINICAL SIGNS

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS

The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In this study STIMULATION INDICES of 1.1, 0.5 and 1.0 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v).
Based on these results, the test item was found to be a non-sensitizer when tested at the described concentrations in ethanol:water, 7:3 (v/v).
Executive summary:

In order to study a possible allergenic potential of the test item three groups of four female mice each were treated with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle (ethanol:water, 7:3 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine eH-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were washed subsequently and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a 13- scintillation counter.

No test item-related clinical signs were observed .

All treated animals survived the scheduled study period.

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX (S.I.).

Group 2 test item concentration = 2.5 % (w/v); SI= 1.1

Group 3 test item concentration = 5 % (w/v) ; SI =0.5

Group 4 test item concentration = 10 % (w/v); SI = 1.0

No linear increase of S.l. in relation to the concentrations applied could be observed which might be related to the limited penetration ratio of the test item dilution.

CONCLUSION

In this study STIMULATION INDICES of 1.1, 0.5 and 1.0 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v).

A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX. Based on these criteria, the test item was found to be a non-sensitizer when tested at the described concentrations in ethanol:water, 7:3 (v/v). 10

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In this study STIMULATION INDICES of 1.1, 0.5 and 1.0 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v). A test item is regarded as a sensitizer in the LLNA if the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the STIMULATION INDEX. Based on these criteria, the test item was found to be a non-sensitizer when tested at the described concentrations in ethanol:water, 7:3 (v/v).


Migrated from Short description of key information:
In order to study a possible allergenic potential of the test item three groups of four female mice each were treated with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v) in ethanol:water, 7:3 (v/v) by topical application to the dorsum of each ear lobe (left and right) on three consecutive days.
No increase of the stimulation index was recorded.

Justification for selection of skin sensitisation endpoint:
only available study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification

Based on a negative result in an LLNA no classsification is warranted