Registration Dossier

Administrative data

Description of key information

OECD 429; GLP; LLNA in mice; doses: 5%, 10%, 25%; SI below 1.2, no sensitizer (RCC, 2006).

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:
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:
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
Remarks:
RCC Ltd, 4452 Itingen, Switzerland
Type of study:
mouse local lymph node assay (LLNA)
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:
>= 0.8 - <= 1.2
Variability:
SD: 0.3 and 0.7
Test group / Remarks:
all test groups
Remarks on result:
other: see Remark
Remarks:
5 % (w/v) test item: 1.2 ± 0.3 (SD) 10 % (w/v) test item: 1.2 ± 0.7 (SD) 25 % (w/v) test item: 0.8 ± 0.3 (SD) No dose-response relationship was observed. Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 404 - <= 629
Variability:
SD: from 79 to 381
Test group / Remarks:
all tested groups
Remarks on result:
other: Control: 516 ± 79 (SD) dpm /node 5 % (w/v) test item: 604 ± 175 (SD) dpm / node 10 % (w/v) test item: 629 ± 381 (SD) dpm / node 25 % (w/v) test item: 404 ± 169 (SD) dpm / node

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:
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:
read-across source
Parameter:
SI
Value:
>= 0.8 - <= 1.2
Variability:
SD: 0.3 and 0.7
Test group / Remarks:
all test groups
Remarks on result:
other: based on read-across
Parameter:
other: disintegrations per minute (DPM)
Value:
>= 404 - <= 629
Variability:
SD: from 79 to 381
Test group / Remarks:
all tested groups
Remarks on result:
other: based on read-across
Cellular proliferation data / Observations:
SI: 5 % (w/v) test item: 1.2 ± 0.3 (SD) 10 % (w/v) test item: 1.2 ± 0.7 (SD) 25 % (w/v) test item: 0.8 ± 0.3 (SD) No dose-response relationship was observed. Calculation of the EC 3 value was not performed because no test concentrations produced a STIMULATION INDEX (S.I.) of 3 or higher.

DPM: Control: 516 ± 79 (SD) dpm /node 5 % (w/v) test item: 604 ± 175 (SD) dpm / node 10 % (w/v) test item: 629 ± 381 (SD) dpm / node 25 % (w/v) test item: 404 ± 169 (SD) dpm / node
Interpretation of results:
GHS criteria not met
Conclusions:
Based on a LLNA performed with the read-across substance, the target substance was not assumed to be a skin sensitizer.
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). This data is used to evaluate the test article’s potential to cause skin sensitization.

 

In the first study, groups of four mice per dose group were treated with a structural analogue dissolved in DMSO once daily for three consecutive days. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Due to the intense red color of the test item local irritation reactions such as ear redness could not be detected. In this study Stimulation Indices (S.I.) of 1.4, 1.3 and 2.0 were determined with the test item at concentrations of 3.5, 7 and 14% (w/v) in DMSO, respectively. The test item was not a skin sensitizer in this assay (CCR, 2005).

 

In the second study performed with another category member presented as read-across entry, three groups each of five female mice were treated daily with the test item 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. 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. 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) (RCC, 2006).

 

In the study performed with the third read across compound, groups of 6 female CBA/Ca mice each were treated with a 30% w/w preparation of the test substance in AOO 4:1 (mixture of acetone:olive oil Ph.Eur./DAB in a ratio 4:1 parts by volume) or with the vehicle alone. No signs of systemic toxicity were noticed. On the2nd and 3rd day of application and on the day of lymph node removal slight black discoloration of the ears were noticed in all animals of the test group. The test substance did not induce a statistically significant or biologically relevant response of the auricular lymph nodes when applied as a 30% preparation (highest applicable concentration) in AOO 4:1. The limited, but statistically significant increase in ear weight indicates some irritation of the ear at this concentration. In conclusion, the test article does not have a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen (BASF, 2005).

 

In a fourth study with another category member groups of four male CBA/Ca mice per dose level were treated with a preparation of the test substance in propylene glycol or with the vehicle alone. Each test animal was applied with 25 µL of the test substance preparation to the dorsum of both ears for three consecutive days. Lymph node response was evaluated by measuring the proliferative capacity of the prepared lymph node cells. The application of the test substance at concentrations of 3%, 10% and 30% w/v in propylene glycol resulted in an increase in isotope incorporation which was less than 3-fold at all three concentrations (SI indices were: 1.16 (3%); 1.61 (10%); 1.31 (30%)). Therefore, the test substance is considered to be not sensitizing (CTL, 1999).

 

Conclusion: No data generated with the test article are available. But according to the category approach, the data available for other category members is used to assess the sensitization potential of the test article. Based on the available and reliable data obtained in Local Lymph Node Assays with four category members, no classification for sensitization is warranted. The SI indices for the tested substances were all below 3. Therefore, the test article is characterized as not sensitizing based on this read across/category approach.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for sensitization is not warranted under Regulation (EC) No.1272/2008.