4-(1,1-dimethylethyl)cyclohexyl acrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.04. - 24.05.2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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:

4-(1,1-dimethylethyl)cyclohexyl acrylate

EC Number:

282-104-8

EC Name:

4-(1,1-dimethylethyl)cyclohexyl acrylate

Cas Number:

84100-23-2

Molecular formula:

C13H22O2

IUPAC Name:

4-tert-butylcyclohexyl prop-2-enoate

Test material form:

liquid

Remarks:

colorless, clear

In vivo test system

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0.1, 0.5, 2 and 5%

No. of animals per dose:

5 (female)

Details on study design:

Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 0.1, 0.5, 2, and 5% in MEK. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79.1 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance.

Positive control substance(s):

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

Statistics:

Where appropriate, the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
All calculations conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count were performed with validated program R Script STABW-mitStat.Rnw).
Within the program a statistical analysis conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. Statistical significance was set at the five per cent level (p < 0.05). Additionally, the Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers. No outliers were detected.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

0.1% test substance

Results 2

Key result

Parameter:

SI

Value:

6.7

Test group / Remarks:

2% test substance

Results 3

Key result

Parameter:

SI

Value:

23

Test group / Remarks:

5% test substance

Results 4

Parameter:

EC3

Value:

1

Any other information on results incl. tables

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant and biologically relevant increase in lymph node weights and –cell counts was observed in the two highest test item groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count of the two highest test item groups exceeded this threshold.

The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not exceeded in any of the treated groups.

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)^a)	SD	S.I.
Vehicle Control Group (MEK)	1040.1	784.8	1.0
0.1% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	1250.5	617.6	1.2
0.5% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	1042.1	341.0	1.0
2% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	6990.5	1039.8	6.7^S
5% 2-Propenoic acid, 4-(1,1-dimethylethyl)cyclohexyl ester	24055.1	10231.8	23.1^S

^a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

^S      statistically significant (p < 0.05)

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 3	0.5 (a)	1.0 (b)
Test Group 4	2 (c)	6.7 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 1% (w/w)

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

Applicant's summary and conclusion

Interpretation of results:

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