Reaction mass of (2R,5R)-2-isopropyl-5-methylcyclohexanone and (2S,5R)-2-isopropyl-5-methylcyclohexanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

other: component of reaction mass

Adequacy of study:

key study

Study period:

from 2012-04-11 to 2012-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted under GLP in accordance with OECD TG 429.

Data source

Reference

Reference Type:

secondary source

Title:

No information

Author:

ECHA disseminated dossier

Year:

2012

Bibliographic source:

Dossier CAS 14073-97-3

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

- Name of test material (as cited in study report): 620103 Menthone / Iso Menthone racemic

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories BV, Maasheseweg 87c, 5804 AB Venray / Netherlands
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 18.0-24.4 g
- Housing: group housing
- Diet (e.g. ad libitum): Teklad Global Rodent 2914C – Rezeptur 3255 GLP Batch 73/11 (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland), available ad libitum.
- Water (e.g. ad libitum): Community tap water from Itingen/Switzerland, available ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.5-20.4
- Humidity (%): 37.4-62.2
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

25, 50 and 100 % (w/v) in vehicle

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The highest test item concentration which can be technically used was a 75% (w/w) solution in acetone:olive oil (4:1 v/v). As the test item is a well pipettable liquid, it was also tested neat (100%).
- Irritation: At the tested 50 and 100 % concentrations, the animals did not show any signs of irritation.
- Lymph node proliferation response: is expressed as the number of radioactive disintegrations per minute per lymph node (dpm/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of the test groups relative to that recorded for the control group (Stimulation Index S.I.). Before dpm/lymph node values were determined, the mean scintillation-background dpm was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Topical application / Administration of 3HTdR / Determination of incorporated 3HTdR
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:
- First, the exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the S.I.
- Second, the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: Test item preparation was made freshly before each dosing occasion, no more than 4 hours prior to application to the ears.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For the body weight mean values and standard deviations were calculated.

Results and discussion

Any other information on results incl. tables

The following results were obtained for 620103 Menthone / Isomenthone using acetone:olive oil (4:1 v/v) as vehicle:

Test item concentration % (w/v)	Groups	Measurement dpm	Calculation			Result SI
			dpm-BG	Number of lymph nodes isolated	dpm / lymph node^a)
--	BG I	3	--	--	--	--
--	BG II	0	--	--	--	--
--	Group 1	1114	1113	8	139	--
25	Group 2	972	971	8	121	0.9
50	Group 3	4760	4759	8	595	4.3
100	Group4	3547	3546	8	443	3.2

BG = Background (1 mL 5 % trichloroacetic acid) in duplicates

SI = Stimulation Index (Ratio = dpm/lymph node test item treated group / dpm/lymph node Group 1)

a) = Since the lymph nodes of the animals of a dose group were pooled, dmp/lymph node was determined by dividing the measured value by the number of lymph nodes pooled.

Calculation of the EC3 Value

	Test item concentration % (w/v)	SI
TG 1	25 (a)	0.9 (b)
TG 2	50 (c)	4.3 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 40.6 %

EC3 = Estimated concentration for a SI of 3

a, b, c, d = Co-ordinates of the two pairs of data lying immediately above and below the SI value of 3 on the LLNA dose response plot

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item 620103 Menthone/Isomenthone was found to be a skin sensitizer.

Executive summary:

In order to study the possible contact allergenic potential of 620103 Menthone/Isomenthone, three groups of four female mice each were treated daily with the test item at concentrations of 25%, 50% (w/v) in acetone:olive oil (4:1 v/v) and 100% by topical application to the dorsum of each ear lobe for three consecutive days. A control group of four female mice was treated with the vehicle acetone:olive oil (4:1 v/v) only.

Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine, 3HTdR). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes were excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period. Neither clinical signs on the ears of the animals nor systemic findings were observed during the study period.

The results obtained [Stimulation Index (S.I.)] are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

	Test item concentration % (w/v)	SI
Group 2	25	0.9*
Group 3	50	4.3*
Group 4	100	3.2
EC3: 40.6 %

A dose-response relationship was observed. A calculation of the EC3 value was performed using the indicated concentrations (*).

In conclusion, the test item 620103 Menthone/Isomenthone was found to be a skin sensitizer and an EC3 value of 40.6% (w/v) was derived.