4-tert-butylcyclohexanone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation

Remarks:

in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 Apr - 10 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

updated 2010

Deviations:

yes

Remarks:

measurement of cell proliferation by cell counting instead of radioactive labeling; the acute inflammatory skin reaction is determined to discriminate specific from non-specifc activation of immune competent cells in draining lymph nodes.

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted in 1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

adopted in 203

Deviations:

no

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: CPMP/SWP/2145/00 adopted in 2001

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-tert-butylcyclohexanone

EC Number:

202-678-5

EC Name:

4-tert-butylcyclohexanone

Cas Number:

98-53-3

Molecular formula:

C10H18O

IUPAC Name:

4-tert-butylcyclohexan-1-one

Details on test material:

- Name of test material (as cited in study report): 4-tert-Butylcyclohexanone
- Physical state: solid (liquid after melting)
- Analytical purity: 99.58%
- Lot/batch No.: CHHDKP1901

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Nederland, Horst, Netherland
- Age at study initiation: 6 weeks (females)
- Weight at study initiation: 26 - 34 g (females)
- Housing: adaptation period: up to 8 mice were housed together in conventional Macrolon type III cages. Study period: the animals were single-housed in type II cages. Low-dust wood shaving were used as bedding.
- Diet: PROVIMI KLIBA SA 3883 maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

2, 10, and 50%

No. of animals per dose:

6 (females)

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The measurement of cell proliferation was performed by cell counting instead of radioactive labeling. The measurement of the ear swelling after treatment was included as well. By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN cell counts / LN weights) with the immediate unspecific acute skin reaction ( ear swelling / ear weigh) it is possible to discriminate the irritant potential from the sensitizing potential of the compound tested.
- Criteria used to consider a positive response: The stimulation index was calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulation effect the index is always about 1.00 (+/- standard deviation), and the indices of vehicle animals are set to 1.00 (+/- standard deviation).
The "positive level" of the stimulation index was set at 1.4. The "positive level" of ear swelling was set at 2x10* -2 mm increase.
TREATMENT PREPARATION AND ADMINISTRATION: The test item in the formulation, the positive control in the formulation or the vehicle were applied (25 µL/ear), epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2, and d3).

Positive control substance(s):

other: alpha hexyl cinnamic aldehyde

Statistics:

The values from treated groups were compared with those from control group(s; vehicle) by one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to homogeneity like Cochran’s test. Alternatively, if the variances are considered heterogeneous (p ≤ 0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5%. Two sided multiple test procedures were done according to Dunnet or Bonferrono-Holm, respectively. Outlying values in LN weight were eliminated at a smallest significant differences in the means were calculated by Schaffe’s Method, which according to Sachs can be used for both equal and unequal sizes.

Results and discussion

Positive control results:

The last reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentration of 3%, 10% and 30% clearly showed the sensitizing potential of the test item.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The stimulation indeces were found to be 1.00 (vehicle control), 1.03 (2% treatment group), 1.12 (10% treatment group), 1.24 (50% treatment group) and 1.42 (positive control group).

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: not applicable

Any other information on results incl. tables

Table 1. Ear swelling (NMRI mice, female, 6 animals/group, in 0.01 mm)

Groups	day 1	day 4	Index day 4
1	17.50± 2.98	18.25± 4.13	1.00
2	17.25± 3.60	17.92± 5.03	0.98
3	17.58± 2.93	18.25± 4.13	1.00
4	17.25± 4.37	17.58± 3.80	0.96
5	17.58± 3.80	22.33 *± 12.71	1.22

*= statistically significant increase (p≤0.05)

Table 1. Ear weight (NMRI mice, female, 6 animals/group, in mg per 8 mm diameter punch)

Groups	day 4	Index day 4
1	12.57± 5.00	1.00
2	12.73± 7.82	1.01
3	12.65± 5.76	1.01
4	11.90± 6.61	0.95
5	15.94*± 16.98	1.27

*= statistically significant increase (p≤0.05)

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified