[No public or meaningful name is available]

Endpoint summary

Description of key information

One reliable (Klimisch 1) sensitisation study was available for Cyclohexanol, 4-C11-12-alkyl, branched. The sensitising properties of Cyclohexanol, 4-C11-12-alkyl, branched to mice, assessed as a Local Lymph Node Assay (LLNA) was reported by Lütkenhaus K., (2012) in an OECD 429 guideline and GLP compliant study.

Based on the results of the prescreen test the test item Cyclohexanol, 4 -C11 -12 -alkyl, branched was assessed for sensitising properties at concentrations of 6.25%, 12.5% and 25% (v/v), each diluted with AOO (4:1(v/v) acetone/olive oil). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed. 5 mice per test and control group were examined.

Two of three tested concentrations exceeded the stimulation index of 3 (SI = 2,1 at 6.25%, SI = 3.7 at 12.5% and SI = 9.3 at 25% test substance concentration). The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 9.72%.

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:

August - November 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP comppliant

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsD

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 17 - 22g
- Housing: in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum
- Water: tap water, sulphur acidified to a pH of approx. 2.8 ad libitum (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Vehicle:

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

Concentration:

Ear thickness prescreen test: 100%, 50%, 25%
Main test: 6.25%, 12.5% and 25%

No. of animals per dose:

Prescreen test: 2 for test substance groups, 1 for control group
Main test: 5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: soluble in vehicle
- Irritation: Animals treated with 100% (undiluted) and 50% test substance showed sticky fur from day 2 until day 6, erythema grade 1 from day 4 until day 6 and slight eschar formation on days 5 and 6. In the animals treated with 25% test substance, wet fur at the application sites was observed on days 2 and 3. No signs of systemic toxicity were detected (including cageside observations of spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoe, changes in the skin and fur, eyes and mucous membranes, salivation and discharge)
- Lymph node proliferation response: no lymph node proliferation assessment performed in the prescreen test

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay
- Criteria used to consider a positive response: Stimulation Index equal to or greater than 3.0

TREATMENT PREPARATION AND ADMINISTRATION:
- Clinical observation: prior to application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application
- Weight assessment: animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR)
- Dose groups: 3 test groups (6.25, 12.5 and 25%) and 1 negative control group (vehicle: AOO, 4:1 (v/v) acetone/olive oil), 5 animals per group
- Test regime:
Topical application: application of 25 µl of the selected solution to the entire dorsal surface of each ear, performed once daily over 3 consecutive days
Administration of 3H-methyl thymidine: dosing with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL 3H-methyl thymidine, diluted to a working concentration of 80 µCi/mL 5 days after the first topical application
Preparation of cell suspension: 5 hours after 3H-methyl thymidine test animals were sacrificed by cervical dislocation, the draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal), cell suspensions of lymph node cells were prepared according to guideline, transferred into scintillation vials and stored at room temperature overnight
Determination of incorporated 3H-methyl thymidine: measurement in a ß-counter and expressed as number of disintegrations per minute (DPM), background 3H-methyl thymidine levels also measured (5% trichloroacetic acid), determination of radioactivity performed individually for each animal

Positive control substance(s):

other: P-Phenylenediammine

Statistics:

Calculation of mean values and standard deviation, test on outliers (Grubbs, Nalimov and Dixon), EC3 values (determined by linear interpolation: EC3 = c+[(3-d)/(b-d)]x(a-c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three)

Positive control results:

Reliability checks were performed on a regular basis in the test laboratory, the recent reliability check performed in October 2012.
Positive control substance: P-Phenylenediammine (CAS 106-50-3, Sigma, puritiy > 98%, Lot 041M0045V) 1%
Vehicle: AOO (4:1 (v/v) acetone/olive oil)
Species/strain: healthy CBA/CaOlaHsd mice
Source: Harlan Winkelmann, Borchen, Germany
Concentrations: 1% on 3 consecutive days
Results: SI = 8.5 ± 2.4

Key result

Parameter:

SI

Remarks on result:

other: Stimulation indices at test substance concentrations (2 of 3 tested concentrations exceeded the stimulation index of 3): 6.25%, SI = 2.1 12.5%: SI = 3.7 25%: SI = 9.3 EC3 = 9.72% (derived by linear interpolation)

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see table below

Table: Radioactive Determination
Test item	Concentration [%]	DPM (mean ± SD)	DPM - mean Background	number of lymph nodes evaluated*	DPM/node (mean ± SD)	SI
Negative control	-	1113.3 ± 417.2	1058.7 ± 417.2	8	529.3 ± 208.6	1.0
Cyclohexanol, 4-C11-12-alkyl, branched	6.25	2306.3 ± 919.3	2251.7 ± 919.3	8	1125.8 ± 459.6	2.1 ± 0.9
Cyclohexanol, 4-C11-12-alkyl, branched	12.5	3972.0 ± 598.7	3917.4 ± 598.7	8	1958.7 ± 299.3	3.7 ± 0.6
Cyclohexanol, 4-C11-12-alkyl, branched	25	9879.8 ± 1260.1	9825.2 ± 1260.1	10	4912.6 ± 630.0	9.3 ± 1.2
Background: Scintillation fluid and TCA	-	54.6 ± 3.8	-	-	-	-
DPM = disintegrations per minute, SD = standard deviation, SI = stimulation index, TCA = trichloroacetic acid
* should be 10 lymph nodes per group, if less some could not be evaluated or were outliers (5 animals per test group, 2 lymph nodes per animal)

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 9.72%. According to the guideline solutions or preparations containing more than 9.72% Cyclohexanol, 4-C11-12-alkyl, branched are expected to have a stimulation index of > 3 and are therefore considered to be dermal sensitiser.

Executive summary:

A sensitisation study (Local Lymph Node Assay) with Cyclohexanol, 4 -C11 -12 -alkyl, branched was performed on mice according to OECD Guideline 429, adopted July 22, 2010.

Based on the results of the prescreen test the test item Cyclohexanol, 4 -C11 -12 -alkyl, branched was assessed for sensitising properties at concentrations of 6.25%, 12.5% and 25% (v/v), each diluted with AOO (4:1(v/v) acetone/olive oil). At the daily clinical observation the animals did not show any visible clinical symptoms and no case of mortality was observed. 5 mice per test and control group were examined.

Two of three tested concentrations exceeded the stimulation index of 3 (SI = 2,1 at 6.25%, SI = 3.7 at 12.5% and SI = 9.3 at 25% test substance concentration). The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 9.72%.

According to the guideline solutions or preparations containing more than 9.72% Cyclohexanol, 4-C11-12-alkyl, branched are expected to have a stimulation index of > 3 and are therefore considered to be dermal sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the classification criteria of regulation (EC) 1272/2008 Cyclohexanol, 4-C11-12-alkyl, branched has to be classified with Skin sensitization, Subcategory 1B, H317.