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Description of key information

Skin sensitization:

Skin sensitization potential of d-Carvone was evaluated by Local Lymph Node Assay (LLNA) The LLNAs were conducted according to the method described inOECD Guideline 429. Groups of female CBA/CA mice (n= 5) were dosed topically on the dorsum of each ear with 25 ml of test material in 1:3 ethanol :diethyl phthalate. Each group received one of five test concentrations prepared as a w/v%. A concurrent vehicle control group was similarly treated with 1:3 ethanol: diethyl phthalate.

Dosing occurred daily for three consecutive days. The animals received no treatment for two days and on the sixth day after the first application, all mice were injected intravenously via the tail vein with 250 ml of phosphate buffered saline containing 20 µCi of radiolabelled methyl thymidine (3HTdR). Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled . Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200-mesh stainless steel gauze. The cell suspensions were washed three times with phosphate buffered saline and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were then pelleted by centrifugation. The cells were resuspended in 1ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporation of 3HTdR was measured by β- scintillationcounting and expressed as disintegrations per minute (dpm) per lymph node.

The EC3 value obtained from LLNA assay performed to assess the sensitizing potential of d-Carvone was 2650 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin.

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:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed journal
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
To evaluate the skin sensitization potential of D-Carvone by Mouse Local Lymph Node Assay.
GLP compliance:
not specified
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material: d-Carvone- IUPAC name: (5S)-2-methyl-5-(prop-1-en-2-yl)cyclohex-2-en-1-one- Molecular formula: C10H14O- Molecular Weight: 150.22 g/mole- Smiles Notation: C1[C@H](CC=C(C1=O)C)C(C)=C- Inchl: 1S/C10H14O/c1-7(2)9-5-4-8(3)10(11)6-9/h4,9H,1,5-6H2,2-3H3/t9-/m0/s1- Substance type: Organic- Physical state: Colorless to pale-yellow liquid
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
No data availlable
Vehicle:
other: 1:3 ethanol :diethyl phthalate.
Concentration:
25 µl (Each group received one of five test concentrations prepared as a w/v%.)
No. of animals per dose:
Total=5
Details on study design:
MAIN STUDYANIMAL ASSIGNMENT AND TREATMENT- Name of test method: LLNA- Criteria used to consider a positive response: EC 3 value TREATMENT PREPARATION AND ADMINISTRATION: Groups of female CBA/CA mice were dosed topically on the dorsum of each ear with 25 µl of test fragrance in 1:3 ethanol: diethyl phthalate.Each group received one of five test concentrations preparedas a w/v%.
Positive control substance(s):
other:
Key result
Parameter:
SI
Value:
2 650
Variability:
not specified
Test group / Remarks:
5 mice
Remarks on result:
other: Sensitizing to skin
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA: Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200- mesh stainless steel gauze. The cell suspensions were washedthree times with phosphate buffered saline and precipitated overnight at 4°C with 5% w/v trichloroacetic acid (TCA). Thesamples were then pelleted by centrifugation. The cells wereresuspended in 1ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporationof 3HTdR was measured by b-scintillation counting and expressed as disintegrations per minute (dpm) per lymph node for each experimental group.DETAILS ON STIMULATION INDEX CALCULATION : The stimulation index (SI)values were calculated for each dose level. A SI of 3 or more was considered to give a positive response.EC3 CALCULATION : The EC3 valuewas taken as a measure of relative potency
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The EC3 value obtained from LLNA assay performed to assess the sensitizing potential of d-Carvone was 2650 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin.
Executive summary:

Skin sensitization potential of d-Carvone was evaluated by Local Lymph Node Assay (LLNA) The LLNAs were conducted according to the method described in OECD Guideline 429. Groups of female CBA/CA mice (n= 5) were dosed topically on the dorsum of each ear with 25 ml of test material in 1:3 ethanol :diethyl phthalate. Each group received one of five test concentrations prepared as a w/v%. A concurrent vehicle control group was similarly treated with 1:3 ethanol: diethyl phthalate.

Dosing occurred daily for three consecutive days. The animals received no treatment for two days and on the sixth day after the first application, all mice were injected intravenously via the tail vein with 250 ml of phosphate buffered saline containing 20 µCi of radiolabelled methyl thymidine (3HTdR). Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled . Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200-mesh stainless steel gauze. The cell suspensions were washed three times with phosphate buffered saline and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were then pelleted by centrifugation. The cells were resuspended in 1ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporation of 3HTdR was measured by β- scintillationcounting and expressed as disintegrations per minute (dpm) per lymph node.

The EC3 value obtained from LLNA assay performed to assess the sensitizing potential of d-Carvone was 2650 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitization:

In different studies, D-Carvone has been investigated for potential for dermal irritation to a greater or lesser extent. The studies are based on in vivo experiments in rabbits along with human data for target chemical D-Carvone .

Skin sensitization potential of d-carvone was evaluated by Mouse Local Lymph Node Assay , Human maximization test and Human repeated insult patch test (HRIPT) conducted by Anne Marie Api, David Basketter, and Jon Lalko summarized in Cutaneous and ocular toxicology, 34, no. 4, (2015), 298-302.

Mouse Local Lymph Node Assay:

Skin sensitization potential of d-Carvone was evaluated by Local Lymph Node Assay (LLNA) The LLNAs were conducted according to the method described in OECD Guideline 429. Groups of female CBA/CA mice (n= 5) were dosed topically on the dorsum of each ear with 25 ml of test material in 1:3 ethanol :diethyl phthalate. Each group received one of five test concentrations prepared as a w/v%. A concurrent vehicle control group was similarly treated with 1:3 ethanol: diethyl phthalate.

Dosing occurred daily for three consecutive days. The animals received no treatment for two days and on the sixth day after the first application, all mice were injected intravenously via the tail vein with 250 ml of phosphate buffered saline containing 20 µCi of radiolabelled methyl thymidine (3HTdR). Five hours later, the mice were euthanized and the draining auricular lymph nodes were excised and pooled . Suspensions of the lymph node cells were prepared by mechanical disaggregation through 200-mesh stainless steel gauze. The cell suspensions were washed three times with phosphate buffered saline and precipitated overnight at 4 °C with 5% w/v trichloroacetic acid (TCA). The samples were then pelleted by centrifugation. The cells were resuspended in 1ml of TCA and transferred to scintillation vials containing 10 ml of scintillation fluid. The incorporation of 3HTdR was measured by β- scintillationcounting and expressed as disintegrations per minute (dpm) per lymph node.

The EC3 value obtained from LLNA assay performed to assess the sensitizing potential of d-Carvone was 2650 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin.

Human maximization test :

d-Carvone was used as a test material to evaluate it’s skin sensitization potential on humans by human maximization test. d-Carvone used in petrolatum vehicle under occlusion on forearms or back of all subjects for five alternate-day 48 h periods.  Patch sites were pre-treated for 24 h with 10% aqueous sodium lauryl sulfate (SLS) under occlusion

  Following a 10–14 day rest period, challenge patches were applied under occlusion to a fresh forearm site for 48 h. Challenge applications were preceded by 30–60 min SLS treatment. Reactions were read atpatch removal and again at 96 h after the initiation of application. Scoring of the test sites was also carried out at 48, 72 and 96 h post-patching using the scoring scale

  The LOEL value obtained from HTM assay performed to assess the sensitizing potential of d-Carvone was 1376 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin

Human repeated insult patch test (HRIPT):

The HRIPT was performed to evaluate the skin sensitization potential ofd-Carvone on 100 human volunteers. Each HRIPT study received approval from an independent ethical review committee (IRB institutional review board) prior to its initiation. During the induction phase an occlusive webril/adhesive patch (25mm Hill Top Chamber System) was used. 0.3 ml of the test material in 1:3 ethanol:diethyl phthalate (vehicle) was applied to each patch. The test material was allowed to volatilize for at least 15 min but no longer than 40 min prior to application to the skin.

 

The left side of the back was used for the test area during the induction phase. Patches remained in place and were kept dry for approximately 24 h, after which time they were removed. A 24 h period, during which no test materials were applied. For alternate days test sites were observed and scored. The identical test site was then retreated until nine induction applications were completed over a period of approximately 3 weeks A rest period of approximately 2 weeks followed the last induction patch. No test materials were applied during the rest period. At the challenge phase, the original induction test sites were observed and each subject queried as to whether any reaction had been experienced during the rest period. The untreated right side of the back was used for the for the challenge phase.

 

Patches were applied as in the induction phase and held in place for 24 h after which time they were removed and the challenge site scored. The original test sites were also observed. Scoring of the test sites was also carried out at 48, 72 and 96 h

  The NOEL value obtained from LLNA assay performed to assess the sensitizing potential of d-Carvone was 2657 microgram/sq cm. Based on this value, d-Carvone can be considered to be sensitizing to skin.

The above experimental data was supported by experimental result summarized in TOXICOLOGY AND CARCINOGENESIS STUDIES OF d-CARVONE (CAS NO. 2244·16·8) IN B6C3F1 MICE (GAVAGE STUDIES), NTRL, NIH Publication No. 90-2836 Feb for the target d-carvone

d- carvone was used as a test material on human volunteers.d- carvone when applied on human in human maximization test in 5% petrolatum showed positive sensitization potential on humans . Hence, d-carvone was considered as skin sensitizer on human.

The above experimental data was further supported by experimental result summarized in Annex 1 Background document to the Opinion proposing harmonised classification and labelling at Community level of carvone Committee for Risk Assessment RAC –ECHA , adopted on 4 june,20 for the target.

d- Carvone was used as a test material to evaluate skin sensitization potential on 15 guinea pigs by Freund’s complete adjuvant test (FCAT) .In induction treatment d- Carvone was intra-dermally subjected in the concentration 5% in Freund’s complete adjuvant and challenged in the concentration 1% carvone by topical application. the above test produced erythema which was persistant on 13 out of 15 test animal 72 hr after the challenge exposure. Hence, d-Carvone was considered as skin sensitizer on guinea pigs. d- Carvone was used as a test material to evaluate skin sensitization potential on 15 guinea pigs by Freund’s complete adjuvant test (FCAT) .In induction treatment d- Carvone was intra-dermally subjected in the concentration 5% in Freund’s complete adjuvant and challenged in the concentration 1% carvone by topical application. the above test produced erythema which was persistant on 13 out of 15 test animal 72 hr after the challenge exposure. Hence, d-Carvone was considered as skin sensitizer on guinea pigs.

The above experimental data was supported by experimental result summarized in Food and Cosmetics Toxicology,Vol. 16, Pg. 673, 1978 for the target d-Carvone

d-Carvone was used as a test material to evaluate skin sensitization reaction on 25 human volunteers.d-Carvone when applied on the skin of 25 human volunteers in the concentration 2% in petrolatum produced no skin sensitization reactions. Hence, d-carvone is considered to be not sensitizing to skin.

Even though the above result states d-Carvone as not skin sensitizer , this result must be due to the low concentration of d-Carvone used for the test.

Based on the experimental results on skin sensitization potential d-Carvone can be considered sensitizing to the skin and can be classified under the category “Category 1 (skin sensitising) based on GHS criteria” as per CLP regulation.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the experimental results on skin sensitization potential d-Carvone can be considered sensitizing to the skin and can be classified under the category “Category 1 (skin sensitising) based on GHS criteria” as per CLP regulation.