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Toxicological information

Skin sensitisation

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Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 August 2017 - 26 September 2017
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
according to guideline
EU Method B.6 (Skin Sensitisation)
Version / remarks:
according to guideline
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Physical appearance: white powder with lumps
- Test item storage: at room temperature protected from light
Specific details on test material used for the study:
pH (1% in water): 5.12-4.78

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks old
- Weight at study initiation: 19.7-23.9 g
- Housing: group housed (5/cage) in polycarbonate cages containing sterilized sawdust as bedding material
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

- Temperature (°C): 22
- Humidity (%): 46-71
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light):12
- IN-LIFE DATES: From: 30 Aug 2017 To: 25 Sept 2017

Study design: in vivo (LLNA)

5, 10 and 25%
No. of animals per dose:
Details on study design:
- Compound solubility:
- Irritation: At a 50% test item concentration both animals showed a skewed head on Days 1 and 2 and one animal showed ptosis on days 2 and 3. Scaliness was noted on the ears of both animals on day 6. At a 25% test item concentration scaliness was noted on one ear of one animal at day 6. No further signs of irritation were observed.
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on days 1 and 3, and on day 6. Variations in ear thickness for all pre-screen animals during the observation period were less than 25% from Day 1 pre-dose values (i.e. between 2 and 20%).
- Erythema scores: 0 for both concentrations tested.
- Based on the clinical signs indicative of systemic toxicity of the test item, the highest test item concentration selected for the main study was a 25% concentration.

Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle:
- Induction (days 1, 2 and 3): The dorsal surface of both ears was topically treated with the test item (25 μL/ear). The control animals were treated in the same way but with the vehicle instead of the test item.
- Excision of the nodes (day 6): Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20%. 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 PBS.
- Tissue processing for radioactivity (day 6): Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). 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) and then stored in the refrigerator until the next day.
- Radioactivity measurements (day 7): Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactivity 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 came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

- Mortality: twice daily
- Postdose observations: once daily on days 1-6 (on days 1-3 between 3 and 4 hours after dosing)
- Body weights: on day 1 and day 6 (individually weighed)
- Irritation: once daily on days 1-6 (on days 1-3 within 1 hour after dosing)
- Necropsy: no necropsy was performed since all animals survived until the end of the observation period.

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.

If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The SI values calculated for the item concentrations 5, 10 and 25% were 1.2, 2.3 and 5.6 respectively. An EC3 value of 13.2% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 14.1, 17.3, 9.8, 17.8, 18.0 and 14.7%.

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
Key result
Test group / Remarks:
Key result
Test group / Remarks:
Cellular proliferation data / Observations:
No erythema was observed in any of the animals. White test item remnants were present on the dorsal surface of the ears of all test item treated animals between Days 2 and 6, which did not hamper scoring of the skin reactions.
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.
No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 509, 764 and 714 DPM, respectively. The mean DPM/animal value for the vehicle control group was 540 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 0.9, 1.4 and 1.3, respectively.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Not classified according to Regulation (EC) No. 1272/2008
Since there was no indication that the test item elicits a SI ≥ 3 when tested up to 25%, ROC 118 was not considered to be a skin sensitizer. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).
Executive summary:

A Local Lymph Node Assay was performed, according to OECD guideline 429 and GLP principles, to asses the skin sensitising potential of ROC-118. Three groups of five female mice were treated with test item concentrations of 5, 10 or 25% w/w, these concentrations were selected based on the results of a pre-screen test. Five vehicle control animals were similarly treated with the vehicle (dimethylformamide) alone.

The test item was applied on three consecutive days, by open application on the ears of the animals. Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. 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 calculated for each concentration group. All auricular lymph nodes of the animals of both the treatment groups and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals. The SI values calculated for the test item concentrations 5, 10 and 25% were 0.9, 1.4 and 1.3, respectively. Since the test item did not induce a SI>3 when tested up to 25%, it is not considered to be a skin sensitizer. ROC-118 is not classified according to GHS and Regulation (EC) No. 1272/2008.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicated that the test system functioned properly.