Registration Dossier

Administrative data

Description of key information

Skin sensitisation (LLNA, similar to OECD 429, mice): not sensitising

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:
05 - 10 Mar 2014 (preliminary study); 12 - 19 Mar 2014 (main study)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted in July 2010
Deviations:
yes
Remarks:
No measurement of ear thickness (preliminary test)
GLP compliance:
no
Remarks:
The study was not conducted in accordance with GLP since it was performed as internal study for workers safety in factory.
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA:J
Remarks:
CBA/JCrlj
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Japan Inc.
- Age at study initiation: 8 weeks (preliminary study); 9 weeks (main study)
- Weight at study initiation: 20.55 - 22.21 g (preliminary study); 20.13 - 23.85 g (main study)
- Housing: 3 animals each were housed in suspended aluminium cages with stainless steel wire-mesh front and floor (176 x 302 x 130 mm). Cages and aluminium feeders were replaced once a week with washed and sterilized ones.
- Diet: CRF-1 (Oriental Yeast Co., Ltd., Japan), ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: 1 - 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): More than 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1, 5 and 25% (w/v)
No. of animals per dose:
1 (preliminary study), 3 (main study)
Details on study design:
PRE-SCREEN TESTS: 1 female mouse per dose group was treated by daily application of 25 μL of the undiluted test substance to the dorsal surface of the ear, for 3 consecutive days
- Compound solubility: Soluble in acetone/olive oil (4:1 v/v) at 25% (w/v)
- Irritation: The irritation for ears was evaluated on Days 1, 2 and 5
- Systemic toxicity: Observation for clinical signs was performed once daily during the experimental period; body weights were measured on day 0 and day 5; draining auricular lymph nodes from both ears were excised and weighed on day 5

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Incorporation of 3H-methyl thymidine in draining lymph nodes
- Criteria used to consider a positive response: Determination of cellular proliferation (incorporated radioactivity as disintegrations per minute (DPM)) and the stimulation index

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test material was applied to the entire dorsal surface of each ear of each mouse. The application was performed once daily for three consecutive days. The irritation (erythema) for ears was evaluated on Days 1, 2 and 5. On Day 5, all mice received a 250 µL intravenous injection containing 2.96 MBq (80µCi)/mL of Methyl-3H-thymidine diluted in phosphate-buffered saline (PBS) via the lateral tail vein. Five hours later, the animals were sacrificed and auricular lymph nodes from both ears were excised and pooled per each dose group and lymph node cells were collected. Incorporated radioactivity was measured by scintillation counting. Observation for clinical signs was performed once daily during the experimental period. The body weights of the mice were measured Day 0 and the last day of the experiment (Day 5).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Mean body weight values and mean lymph nodes weights were calculated.
Positive control results:
The positive control (25% hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v)) led to a SI of 11.2, thus meeting the reliability criteria for the LLNA (SI ≥ 3).
Key result
Parameter:
SI
Value:
1.33
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
1.1
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
0.577
Test group / Remarks:
25%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION
The SI of the 1, 5 and 25% treatment group was 1.33, 1.1 and 0.577, respectively. None of the test substance concentrations produced as 3-fold increase in 3HTdR incorporation.

EC3 CALCULATION
None of the SI values were above 3 and it is therefore not possible to determine a EC3 concentration.

CLINICAL OBSERVATIONS
There was no effect on clinical signs in any dose group.

BODY WEIGHT
Body weight gains were unaffected in all dose groups.

Table 1: Results of the preliminary study

Concentration (%) Body weight (g) Irritation response Lymph nodes weight (g)
Prior D5 Gains D1 D2 D5
1 21.74 23.07 1.33 n.e. n.e. n.e. 0.0039
5 20.55 22.18 1.63 n.e. n.e. n.e. 0.0038
25 22.21 24.01 1.80 n.e. n.e. n.e. 0.0050

D = day

n.e. = no erythema

Table 1: Results of the main study

Concentration (%) Body weight (g) Irritation response Lymph nodes weight (g) Radioactivity incorporated
  Prior D5 Gains D1 D2 D5 Individual Sum Average DPM SI
Control 21.16 42483 1.88 n. e. n. e. n. e. 0.0062 0.0151 0.005 1082 -
23.07 23.89 0.82 0.0048
20.71 22.23 1.52 0.0041
1 23.85 25.17 1.32 n. e. n. e. n. e. 0.0060 0.0164 0.0055 1437 1.33
21.36 22.10 0.74 0.0060
21.63 22.57 0.94 0.0044
5 22.47 23.49 1.02 n. e. n. e. n. e. 0.0064 0.0153 0.0051 1189 1.10
21.31 22.56 1.25 0.0045
21.75 22.86 1.11 0.0044
25 21.35 22.48 1.13 n. e. n. e. n. e. 0.0047 0.0124 0.0041 624 0.577
22.78 22.81 0.03 0.0033
20.13 20.39 0.26 0.0044
Positive control not determined 12079 11.2

D = day

n.e. = no erythema

DPM = disintegrations per minute

SI = stimulation index

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation(EC) No. 1272/2008
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The skin sensitising properties of the registered substance (CAS 61167-58-6) were tested in a study performed similar to OECD guideline 429 using the murine local nymph node assay (Environmental Health Science Laboratory, 2014). Groups of three mice were exposed daily, for three consecutive days, to 1, 5 and 25% of the test substance or to the vehicle alone, on the dorsum of both ears. Subsequently, mice were injected intravenously with [3H]-thymidine. Radioactivity was measured as a function of isotope incorporation in draining auricular lymph nodes. The test substance (CAS 61167-58-6) did not induce a dose-dependent thymidine incorporation. Thus, test concentrations of 1, 5 and 25% resulted in SI values of 1.33, 1.10 and 0.577, respectively. The positive control (25% hexyl cinnamic aldehyde in acetone/olive oil (4:1 v/v)) resulted in a SI (= 11.2), thus meeting the reliability criteria for the LLNA (SI ≥ 3).Therefore, the test substance (CAS 61167-58-6) is considered to be a non-sensitizer in the conducted LLNA test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation with the test substance (CAS 61167-58-6) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.