Registration Dossier

Administrative data

Description of key information

The SI values calculated for AC-6000 at concentrations of 25, 50, and 100% were 1,8, 1.3 and 1.2 respectively. There was no indication that the test substance could elicit an SI ≥ 3 when tested up to 100%, therefore; the test substance should not be considered a skin sensitiser.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
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)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
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: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: 12 weeks
- Weight at study initiation: 20-26 g
- Housing: individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France)
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten, GbmH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2-23.7
- Humidity (%): 39-65
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100%
No. of animals per dose:
5
Details on study design:
The highest substance concentration selected for the main study (100%) was based on the results of a preliminary irritation study in which no irritation was observed in any of the animals examined.

Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.
The dorsal surface of both ears was epidermally treated (25 µl/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.
Three days after the last exposure each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 µCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate® (0.2 ml/animal). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) at 4ºC during the night.
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
The SI values calculated for the substance concentrations 5, 10 and 25% were 1.3, 1.5 and 5.5, respectively. An EC3 value of 15.6% was calculated using linear interpolation.
Key result
Parameter:
SI
Value:
1.8
Test group / Remarks:
25% concentration
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/v) Stimulation index Result 0 1.0 - 25 1.8±0.5 negative 50 1.3±0.4 negative 100 1.2±0.2 negative
Key result
Parameter:
SI
Value:
1.3
Test group / Remarks:
50% concentration
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
100% concentration
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/v) Disintegrations per minute 0 226±43 25 400±85 50 291±56 100 264±22

Skin reactions / irritation

No skin irritation was observed in any of the animals examined.

Macroscopy of the auricular lymph nodes and surrounding area

All nodes of the experimental and control groups were considered normal in size. no macroscopic abnormalities of the surrounding area were noted.

Body weights

No significant effects were reported.

Toxicity and mortality

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Interpretation of results:
GHS criteria not met
Conclusions:
The SI values calculated for the substance concentrations 25, 50, and 100% were 1.8, 1.3 and 1.2 respectively. There was no indication that the test substance could elicit an SI ≥ 3 when tested up to 100%, therefore; the test substance should not be considered a skin sensitiser.
Executive summary:

The skin sensitising properties of AC-6000 were determined in a GLP study according OECD guideline 429. For this purpose, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control (Acetone / Olive oil) animals were similarly treated. Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No skin irritation was observed in any of the animals examined. All nodes of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 400, 291 and 264 respectively. The mean DPM/animal value for the vehicle control group was 226. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 1.3 and 1.2 respectively. There was no indication that the test substance could elicit an SI ≥ 3 when tested up to 100%. Based on these results AC-6000 should not be considered a skin sensitiser.

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

The skin sensitising properties of AC-6000 were determined in a GLP study according OECD guideline 429 (Local Lymph Node Assay (LLNA)) (NOTOX B.V., 2007). For this purpose, three groups of five experimental animals were treated with test substance concentrations of 25%, 50% or 100% on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Acetone / Olive oil). Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of Disintegrations Per Minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

No skin irritation was observed in any of the animals examined. All nodes of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 400, 291 and 264 respectively. The mean DPM/animal value for the vehicle control group was 226. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.8, 1.3 and 1.2 respectively. There was no indication that the test substance could elicit an SI ≥ 3 when tested up to 100%. Based on these results AC-6000 should not be considered a skin sensitiser.


Short description of key information:
The substance was not a skin sensitiser in a local lymph node assay in the mouse.

Justification for classification or non-classification

Based on the results of the skin sensitisation study, classification according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 is not needed.