Registration Dossier

Administrative data

Description of key information

1,1-dimethylpropyl 1-methoxycyclohexyl peroxide is not a skin sensitizer.

The assessment of contact hypersensitivity of 1,1-dimethylpropyl 1-methoxycyclohexyl peroxide (LUPEROX V10) was performed in the Mouse (Local Lymph Node Assay) following the OECD guideline No.429 (2010) (Latour, 2015). Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). 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 subsequently calculated for each group. The very slight irritation of the ears as shown by all animals treated at 50% and 100% on Day 3 was considered not to have a toxicologically significant effect on the activity of the nodes. 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 substance concentrations 25, 50 and 100% were 819, 1079 and 1209 DPM, respectively. The mean DPM/animal value for the vehicle control group was 500 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.6, 2.2 and 2.4, respectively. Since there was no indication that the test substance elicited a SI = 3 when tested up to 100%, LUPEROX V10 was not considered to be a skin sensitizer.

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 guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: 19-22 g
- Housing: in group in labeled Makrolon cages (MIII type; height 18 cm)
- Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany, ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 25, 50, 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: soluble in Acetone/Olive oil (4:1 v/v)
- Irritation:
Two test substance concentrations were tested; a 50% and 100% concentration. Very slight erythema was noted for all animals on Day 3. No signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mouse local lymphnode assay (LLNA)
- Criteria used to consider a positive response:
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 DPM/vehicle control group.
If the results indicate a SI = 3, the test substance may be regarded as a skin sensitizer.

TREATMENT PREPARATION AND ADMINISTRATION:
. Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 µL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.
The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.
. Excision of the nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 µCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).
After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). 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.
. 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 (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) (Merck, Darmstadt, Germany) and 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. 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 came 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)
Key result
Parameter:
SI
Remarks:
25%
Value:
1.6
Variability:
+/- 0.4
Key result
Parameter:
SI
Remarks:
50%
Value:
2.2
Variability:
+/- 0.5
Key result
Parameter:
SI
Remarks:
100%
Value:
2.4
Variability:
+/- 0.5

Skin reactions / Irritation

The very slight irritation of the ears as shown by all animals treated at 50% and 100% on Day 3 was considered not to have a toxicologically significant effect on the activity of the nodes.

Systemic toxicity

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.

Macroscopy of the auricular lymph nodes and surrounding area

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.

Radioactivity measurements and SI values (Table 4, Figure 1)

Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 819, 1079 and 1209 DPM, respectively. The mean DPM/animal value for the vehicle control group was 500 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.6, 2.2 and 2.4, respectively.

Interpretation of results:
GHS criteria not met
Conclusions:
Since there was no indication that the test substance elicited a SI . 3 when tested up to 100%, LUPEROX V10 was not considered to be a skin sensitizer.
Executive summary:

The assessment of contact hypersensitivity of LUPEROX V10 was performed in the Mouse (Local Lymph Node Assay) following the OECD guideline No.429 (2010). Test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 25, 50 or 100% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)). 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 subsequently calculated for each group. The very slight irritation of the ears as shown by all animals treated at 50% and 100% on Day 3 was considered not to have a toxicologically significant effect on the activity of the nodes. 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 substance concentrations 25, 50 and 100% were 819, 1079 and 1209 DPM, respectively. The mean DPM/animal value for the vehicle control group was 500 DPM. The SI values calculated for the substance concentrations 25, 50 and 100% were 1.6, 2.2 and 2.4, respectively. Since there was no indication that the test substance elicited a SI = 3 when tested up to 100%, LUPEROX V10 was not considered to be a skin sensitizer.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

No classification is warranted according to CLP/GHS criteria.