Registration Dossier

Administrative data

Description of key information

Skin Sensitisation: Not sensitising; OECD 429; Anon. (2017)

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:
24 January - 08 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ca. 2 - 8 ºC in the dark and under nitrogen.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in acetone: olive oil (4: 1 v/v)
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 ºC
- Humidity (%): 30 - 70 %
- Air changes (per hr): At least 15 changes/ hour
- Photoperiod (hrs dark / hrs light): 12 h: 12 h (light: dark)
- IN-LIFE DATES: Not reported
Vehicle:
unchanged (no vehicle)
Remarks:
Acetone: olive oil (4: 1 v/v) was used to prepare 50 and 25 % dilutions
Concentration:
100, 50 and 25 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Soluble
- Irritation: No
- Systemic toxicity: No
- Ear thickness measurements: < 25 % increase
- Erythema scores: No
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non-parametric Kruskal-Wallis Rank Sum and Mann-Whitney U test procedures were used.

Probability values (p) are presented as follows:

P<0.001 ***
P<0.01 **
P<0.05 *
P>0.05 (not significant)
Positive control results:
Concurrent control was within the historical control data. The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.77) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
2.39
Test group / Remarks:
100 %
Parameter:
SI
Value:
2.18
Test group / Remarks:
50 %
Parameter:
SI
Value:
1.04
Test group / Remarks:
25 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: < 25 % at 100 % concentration.

DETAILS ON STIMULATION INDEX CALCULATION: Mean treatment group dpm / mean vehicle dpm

EC3 CALCULATION: n/a

CLINICAL OBSERVATIONS: No signs of systemic toxicity or other clinical signs

BODY WEIGHTS: Within normal limits

Table 1       Stimulation index results

Test Group

Animal #

Individual animal DPMa

Mean group DPM

(std. dev.)

Stimulation index

(SI)

Solvent control

(acetone: olive oil 4:1 v/v)

1-1

2439.33

2647.93

(± 494.05)

n/a

1-2

1979.36

1-3

3323.90

1-4

2814.40

1-5

2682.68

100 % test item

2-1

2418.63

2765.62

(± 1419.40)

1.04

2-2

1253.57

2-3

2248.76

2-4

2821.62

2-5

5085.50

50 % test item

3-1

5282.53

5771.17**

(± 1259.83)

2.18

3-2

3837.39

3-3

7016.92

3-4

6108.25

3-5

6610.76

25 % test item

4-1

6484.02

6337.30**

(±715.81)

2.39

4-2

6983.00

4-3

5108.28

4-4

6606.98

4-5

6504.24

Positive control

(25 % v/v in acetone: olive oil 4:1 v/v)

5-1

9531.50

12624.09**

(± 6427.06)

4.77

5-2

8968.44

5-3

6686.66

5-4

15321.04

5-5

22612.79

** p < 0.01

a2 lymph nodes per animal

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitiser under the conditions of the test. Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.
Executive summary:

OECD 429 (2017) - In a dermal sensitization study with Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene in acetone: olive oil (4:1 v/v)young female adult mice (CBA/Ca (CBA/CaOlaHsd)) were tested using the Local Lymph Node Assay (LLNA)

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100 %, this concentration was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in the vehicle at concentrations of 50 % or 25 % v/v. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity, local skin irritation observed and ear thickness were within normal range. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the studyThe Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

There was no indication that the test item elicits a Stimulation Index ≥ 3 when tested up to 100 %, the test item was considered to be a non-sensitiser under the conditions of the test.

 

In this study, Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was not a dermal sensitizer.

 

Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

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

OECD 429 (2017) - In a dermal sensitization study with Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene in acetone: olive oil (4:1 v/v)young female adult mice (CBA/Ca (CBA/CaOlaHsd)) were tested using the Local Lymph Node Assay (LLNA)

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100 %, this concentration was selected as the highest dose investigated in the main test. Three groups, each of five animals, were treated with 50 μL (25 μL per ear) of the undiluted test item or the test item as a solution in the vehicle at concentrations of 50 % or 25 % v/v. A further group of five animals was treated with the vehicle alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity, local skin irritation observed and ear thickness were within normal range. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the study. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

There was no indication that the test item elicits a Stimulation Index ≥ 3 when tested up to 100 %, the test item was considered to be a non-sensitiser under the conditions of the test.

 

In this study, Reaction mass of (4R)-4-isopropenyl-1-methylcyclohexene and (4R)-4-(2-methoxypropan-2-yl)-1-methylcyclohexene was not a dermal sensitizer.

 

Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The substance does not meet the classification criteria for skin sensitisation under GHS or Regulation (EC) No 1272/2008 (CLP).