Registration Dossier

Administrative data

Description of key information

Skin sensitisation (in vivo): Sensitising (OECD 429/GLP)

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:
02-11-2004 to 09-11-2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: OECD guideline study - GLP. No information about purity of the test substance and no justification of the choice of vehicle was provided.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
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: Jackson Laboratories, Bar Harbour, ME, USA.
- Age at study initiation: 9-11 weeks at start of dosing
- Weight at study initiation: 19-24 g on Day 1
- Housing: Animals were group housed (5 per cage) upon receipt then 4 per cage when assigned to groups in compliance with National Research Council 'Guide for the Care and Use of Laboratory Animals'.
- Diet (e.g. ad libitum): Certified Rodent Chow 7012C ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.2-26.7℃
- Humidity (%): 22-44%
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Vehicle:
other: Diethyl phthalate/ethanol in a 3:1 ratio. No justification provided.
Concentration:
Test substance: 2.5, 5, 10, 25 and 50% w/v in 3:1 Diethyl phthalate/ethanol

No. of animals per dose:
4 animals per dose for test substance
Details on study design:
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: The reliability of this test has been assessed within a six month interval of the study reported here. Positive control study used for comparison: October 2004 (Study number: 0787MR25.001; Table 5). Hexyl cinnamic aldehyde was used as the positive control. The criterion for a positive response is that one of more concentrations of the test substance should elicit a 3-fold or greater increase isotope incorporation relative to the vehicle control group.

TREATMENT PREPARATION AND ADMINISTRATION: Mice were treated on the dorsal surface of each ear, once per day on Days 1, 2 and 3. On day 6, 250μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine (5.0Ci/mmol specific activity) was injected into all test and control mice. Five hours later the animals were killed via CO2 asphyxiation and the lymph nodes were removed and pooled to prepare a single cell suspension. The cell suspensions were then washed twice with PBS and precipitated with 5% trichloroacetic acid (TCA) at 2-8C overnight. Pellets were recovered by centrifugation and re-suspended in 1 mL TCA.
Statistics:
Body weight: ANOVA F-test, Dunnett's test.

EC3 formula: EC3 = c +[(3-d)/(b-d)](a-c) where the data points lying immediately above and below the SI value of 3 have the co-ordinates (a,b) and (c,d) respectively.
Positive control results:
Positive control study used for comparison: There was no positive control study conducted concurrently. However, the reliability of the test system wasconfirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; October 2004 (Study number: 0787MR25.001; Table 5).
Parameter:
SI
Value:
1.67
Test group / Remarks:
2.5%
Parameter:
SI
Value:
1.69
Test group / Remarks:
5%
Parameter:
SI
Value:
2.39
Test group / Remarks:
10%
Parameter:
SI
Value:
4.72
Test group / Remarks:
25%
Parameter:
SI
Value:
25.09
Test group / Remarks:
50%
Parameter:
EC3
Value:
13.93

Table 2: Dermal Irritation scores

Mouse No.  Group Day1 Day 2 Day 3 Day 4 Day 5 Day 6
1 1 Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema Erythema Edema
2 1 0 0 0 0 0 0 0 0 0 0 0 0
3 1 0 0 0 0 0 0 0 0 0 0 0 0
4 1 0 0 0 0 0 0 0 0 0 0 0 0
5 2 0 0 0 0 0 0 0 0 0 0 0 0
6 2 0 0 0 0 0 0 0 0 0 0 0 0
7 2 0 0 0 0 0 0 0 0 0 0 0 0
8 2 0 0 0 0 0 0 0 0 0 0 0 0
9 3 0 0 0 0 0 0 0 0 0 0 0 0
10 3 0 0 0 0 0 0 0 0 0 0 0 0
11 3 0 0 0 0 0 0 0 0 0 0 0 0
12 3 0 0 0 0 0 0 0 0 0 0 0 0
13 4 0 0 0 0 0 0 0 0 0 0 0 0
14 4 0 0 0 0 0 0 0 0 0 0 0 0
15 4 0 0 0 0 0 0 0 0 0 0 0 0
16 4 0 0 0 0 0 0 0 0 0 0 0 0
17 5 0 0 0 0 0 0 0 0 0 0 0 0
18 5 0 0 0 0 0 0 0 0 0 0 0 0
19 5 0 0 0 0 0 0 0 0 0 0 0 0
20 5 0 0 0 0 0 0 0 0 0 0 0 0
21 6 0 0 0 0 1 0 1 0 1 0 0 0
22 6 0 0 0 0 1 0 1 0 1 0 0 0
23 6 0 0 0 0 1 0 1 0 1 0 0 0
24 6 0 0 0 0 1 0 1 0 1 0 0 0

Table 3: Body weights

Group Dose Body weights (g) (mean±sem) Change in body weight (g) (mean ± sem)
Day 1 Day 6
1 - (1) 20.5 ± 0.6 20.8 ± 0.5 0.3 ± 0.3
2 3% 21.5 ± 0.6 21.5 ± 1.0 0.0 ± 0.4
3 5% 21.0 ± 0.7 21.5 ± 0.9 0.5 ± 0.3
4 10% 21.0 ± 1.1 22.5 ± 1.0 1.5 ± 0.5
5 25% 20.8 ± 1.0 22.0 ± 0.9 1.3 ± 0.3
6 50% 20.0 ± 0.4 20.5 ± 0.3 0.5 ± 0.3

(1) Diethyl phthalate/ethanol in a ratio of 3:1

Table 4: Local lymph node assay (test substance)

Group Dose dpm  SI (Test/control ratio) Results (2)
1 - (1) 147 - -
2 2.5% 245 1.67 -
3 5% 248 1.69 -
4 10% 351 2.39 -
5 25% 695 4.72 +
6 50% 3688 25.09 +

(1) Diethyl phthalate/ethanol in a ratio of 3:1

(2) Test/control ration of 3.0 or greater represents a positive result

EC3 = 13.93%

Table 5: Local lymph node assay (positive control)

[study run within a six month interval (October 2004 (Study number: 0787MR25.001)]

Treatment Dose dpm (mean±sem) SI (Test/control ratio) Results (2)
Vehicle (1) - 152 ± 11 - -
HCA 5% 227 ± 45 1.49 -
HCA 15% 428 ± 166* 2.82 -
HCA 35% 524 ± 59** 3.45 +

(1) Diethyl phthalate/ethanol in a ratio of 3:1

(2) Test/control ration of 3.0 or greater represents a positive result

*Statistically sig. difference when the log DPM data of test treated group was compared to vehicle control (p<0.05)

**Statistically sig. difference when the log DPM data of test treated group was compared to vehicle control (p<0.001)

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the criteria of this study, treatment with Acetyl cedrene at 25% and 50% (w/v) resulted in a stimulation index of 3 or greater and hence is consdiered to be a potential skin sensitiser with an EC3 of 13.93.
Executive summary:

In a dermal sensitization study (0787MR25.002) with Methyl Cedryl Ketone (2.5, 5, 10, 25 and 50% w/v in 3:1 Diethyl phthalate/ethanol), young adult female CBA/J mice (4/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the most recent positive control assay (Hexyl cinnamic aldehyde; October 2004 (Study number: 0787MR25.001)

There was no mortality and all animals appeared normal throughout the study. There were no statistically significant differences observed between any treatment groups with respect to body weight. Very slight erythema was noted in all animals treated with acetyl cedrene (50% w/v) on Days 3 -5. Treatment with Methyl Cedryl Ketone at 2.5%, 5%, 10%, 25% or 50% (w/v) resulted in stimulation indices (SI) of 1.67, 1.69, 2.39, 4.72 and 25.09 respectively. The EC3 was calculated to be 13.93%. In this study, Methyl Cedryl Ketone is a potential skin sensitizer.

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

Skin sensitisation (in vitro):

There are no in vitro skin sensitisation tests available

Skin sensitisation (in vivo):

There is one in vivo LLNA assay test conducted in 2005 available.

In a dermal sensitization study (OECD 429/GLP) with Methyl Cedryl Ketone (2.5, 5, 10, 25 and 50% w/v in 3:1 Diethyl phthalate/ethanol), young adult female CBA/J mice (4/group) were tested using the local lymph node assay. The reliability of the test system was confirmed by the most recent positive control assay (Hexyl cinnamic aldehyde). There was no mortality and all animals appeared normal throughout the study. There were no statistically significant differences observed between any treatment groups with respect to body weight. Very slight erythema was noted in all animals treated with acetyl cedrene (50% w/v) on Days 3 -5. Treatment with Methyl Cedryl Ketone at 2.5%, 5%, 10%, 25% or 50% (w/v) resulted in stimulation indices (SI) of 1.67, 1.69, 2.39, 4.72 and 25.09 respectively. The EC3 was calculated to be 13.93%. In this study, Methyl Cedryl Ketone is a potential skin sensitizer.

The LLNA study provided an EC3 value which provides a quantitative basis for determination of the relative skin sensitizing potency of the test substance. The EC3 value giving rise to a 3 fold increase in lymphocyte proliferation was calculated to be 13.93%. As the EC3 value was >2%, the substance is classified as Skin sensitiser Category 1B.

The results from this study are acceptable to use in the human health risk assessment.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the information in the dossier, the substance Methyl Cedryl Ketone (CAS No 32388-55-9) is classified as Skin sensitiser Category 1B (H317: May cause allergic skin reaction) when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.