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Administrative data

Description of key information

Skin sensitization: Read-across, not sensitizing in LLNA, according OECD TG 429, GLP-compliant, 5 %, 10 %, 25 % test substance in acetone/olive oil, mouse, 2006, K1

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Justification for type of information:
see attached justification.
Reason / purpose for cross-reference:
read-across source
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Remarks on result:
other: based on read-across
Parameter:
SI
Value:
1.2
Test group / Remarks:
10%
Remarks on result:
other: based on read-across
Parameter:
SI
Value:
0.8
Test group / Remarks:
25%
Remarks on result:
other: based on read-across
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-OCT-2005 - 25-JAN-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Physical state: Solid, black powder
- Analytical purity: > 99%
- Storage condition of test material: Room temperature
- Expiration date of the lot/batch: 18 November 2020
- CAS No. Cis: 55034-81-6 Trans: 55034-79-2
Species:
mouse
Strain:
other: CBA/CaHsdRcc(SPF )
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd Laboratory Animal Service, Füllinsdorf / Switzerland
- Age at study initiation: 8- 12 weeks (beginning of acclimatization )
- Weight at study initiation: 16 g - 24 g (ordered )
- Housing: Individual in Makrolon type-2 cages with standard softwood bedding.
- Diet: Pelleted standard Kliba 3433, batch no . 39/05 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst) available ad libitum
- Water: Community tap water from Itingen, available ad libitum.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 3°C
- Humidity (%): 30 - 70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
5%, 10% and 25% (based a non-GLP local toxicity pre-test, 25% was the highest technically applicable concentration in the chosen vehicle)


No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
• In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v; A00), ethanol/water (7/3, v/v) and N,N-dimethylformamide (DMF). The pre-test results showed that 25 % in acetone/olive oil (4/1, v/v) was the highest technically applicable concentration. Only lower concentrations suitable for application could be produced with the other vehicles.
• In a non-GLP local toxicity pre-test with the suitable vehicle A00, concentrations were determined in three single animals each treated with one of three different concentrations (5 %, 10 % and 25 %) in both ears on three consecutive days.
After each topical application, the residual test item was found at all dosing sites.

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle acetone/olive oil (4/1, v/v) was quantitatively added. The weight/volume (w/v) dilutions were prepared individually using a magnetic stirrer as homogenizer. Test item formulations were made freshly before each dosing occasion and no more than 4 hours prior to application to the ears. Homogeneity of the test item in the vehicle was maintained until start of treatment using an appropriate homogenizer.
Three groups each of five female mice were treated daily with the test item at concentrations of 5%, 10% and 25% in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days (application volume: 25 µl). 25% was the highest technically applicable concentration in the vehicle. A control group of five mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-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 subsequently washed 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 ß-scintillation counter.

- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled :
First, 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 .).
Second, 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.

DETERMINATION OF EAR THICKNESS
On days 1, 2 and 3 (before application) as well as on day 6 (prior to necropsy), the thickness of both ear (left and right) of each animal was measured using a micrometer.
Any other observation (erythema, colouration, presence of residual test item, crust, etc .) was noted at each time point.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations of body weights and dpm values were calculated.
Dunnett-test was conducted for assessment of the difference significance between the test item groups and the negative control (vehicle) group.
Parameter:
SI
Value:
1.2
Test group / Remarks:
5%
Parameter:
SI
Value:
1.2
Test group / Remarks:
10%
Parameter:
SI
Value:
0.8
Test group / Remarks:
25%

No biological relevant changes in ear thickness occurred. No deaths occurred during the study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. After the first topical application, the residual test item was found at both dosing sites in all mice of the test item groups, persisting for the remainder of the in-life phase of the study. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age.

Statistical Analysis* 
Group Test item concentration dpm/node M (SD) S.I.M (SD) t value Conclusion
CG 1 - 516 (79) - - -
TG 2 5 % (w/v) 604 (175) 1.2 (0.3) 0.61 --
TG 3 10 % (w/v) 629 (381) 1.2 (0.7) 0.78 --
TG 4 25 % (w/v) 404 (169) 0.8 (0.3) 0.77 --

S.I. = Stimulation Index

* Dunnett-test (G = 4, N= 20, t= 2.59)

-- no significant difference at p </= 0.05 (two sides)

No dose-response relationship was observed. Calculation of the EC3 value was not performed because no test concentrations produced a Stimulation Index (S.I.) of 3 or higher.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study STIMULATION INDICES of 1 .2, 1 .2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. Therefore, the test substance was found to be a non-sensitizer.
Executive summary:

In order to study a possible contact allergenic potential of the test article, a GLP compliant Local Lymph Node Assay according to the OECD guideline No. 429 was performed. Three groups each of five female mice were treated daily with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. 25 % was the highest technically applicable concentration in the vehicle. A control group of five mice was treated with the vehicle acetone/olive oil (4/1, v/v) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-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 subsequently washed 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 ß-scintillation counter. All treated animals survived the scheduled study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. After the first topical application, the residual test item was found at both dosing sites in all mice of the test item groups (Groups 2-4), persisting for the remainder of the in-life phase of the study. In this study STIMULATION INDICES of 1 .2, 1 .2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S .I .) of 3 or higher. Based on this result, the test article was found to be a non-sensitizer when tested up to the highest applicable concentration of 25 % in acetone/olive oil (4/1, v/v).

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

No sensitization studies are available for the test substance. However, four GLP-compliant Local Lymph Node Assays have been conducted according to OECD guideline 429 with four category members (see attached category justification). To fill the data gap, a read-across to EC 479-300-2 was performed. 


The skin sensitizing potential of the test substance was assessed using the Murine Local Lymph Node Assay performed according to GLP and OECD Guideline 429 (RCC A32073, 2006). Three groups of 5 female CBA/CaHsdRcc mice were treated daily with 25 µl of the test item at concentrations of 5%, 10% and 25% in acetone/olive oil (4/1, v/v) to the dorsum of each ear lobe for three consecutive days. 25 % was the highest technically applicable concentration in the vehicle. A control group of five mice was treated with the vehicle acetone/olive oil (4/1, v/v) alone. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the auricular lymph nodes were removed and pooled per group. The proliferative capacity of pooled lymph node cells was determined by the incorporation of ³H-methyl thymidine measured in a ß-scintillation counter. No deaths occurred during the study period. Neither clinical / local signs nor other findings were observed in any animals of the control group. About 2 hours after the first topical application, a slight ear erythema was observed at both dosing sites in all mice of the 10 % and 25 % group, persisting for the remainder of the in-life phase of the study. In addition, on the third application day, a slight ear swelling was observed at both dosing sites in one animal of the 5% group and all mice of the 10 % and 25 % group, persisting for a total of two days. The body weight of the animals, recorded prior to the first application and prior to necropsy, was within the range commonly recorded for animals of the strain and age. In this study stimulation indices of 1.2, 1.2 and 0.8 were determined with the test item at concentrations of 5%, 10 % and 25 % in acetone/olive oil (4/1, v/v), respectively. The test item was therefore found to be a non-sensitizer when tested up to the highest applicable concentration.


 


Further toxicological data of category members:


This finding was supported by the data obtained for additional members of the same substance category. In three LLNA assays conducted according to OECD guideline 429 and in compliance with GLP, three additional members of the same category were analyzed for their sensitization potential. None of the tests gave a positive response, therefore all tested substances were considered as not sensitizing.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008


The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No increase in the stimulation index was observed in the LLNA (OECD 429). Therefore, the substance does not require classification as a skin sensitizer under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.