3,3,5-trimethylcyclohexyl methacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 2016 to 17 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OECD Guidelines for Testing of Chemicals No. 429 “Skin Sensitisation: Local Lymph Node Assay”(22 July 2010)

Deviations:

yes

Remarks:

See "Any other information" for details

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008, B.42. “Skin Sensitisation: Local Lymph Node Assay” (Official Journal L 142, 31/05/2008) amended by Commission Regulation (EU) No 640/2012 of 6 July 2012

Deviations:

yes

Remarks:

See "Any other information" for details

Principles of method if other than guideline:

Due to technical reasons, the actual relative humidity range was 23-87 % instead of 30-70 % as it was indicated in the Study Plan. The DPM measurement was performed more than once and the mean results calculated as appropriate. Measurements ended later, consequently the experiment ended later than indicated in the Study Plan. These deviations are considered not to adversely affect the results or integrity of the study.

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:

3,3,5-trimethylcyclohexyl methacrylate

EC Number:

231-927-0

EC Name:

3,3,5-trimethylcyclohexyl methacrylate

Cas Number:

7779-31-9

Molecular formula:

C13H22O2

IUPAC Name:

3,3,5-trimethylcyclohexyl 2-methylprop-2-enoate

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/CaOlaHsd mice
Source: Envigo, San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy
Hygienic level: SPF at arrival; standard housing conditions during the study
Justification of strain: On the basis of OECD Guideline, mice of CBA/Ca or CBA/J strain can be used. Females are used because the existing database is predominantly based on females.
Number of animals: 4 animals / group
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 11 weeks old (age-matched, within one week)
Body weight range at starting: 20.0 – 20.9 grams (The weight variation in animals in the study did not exceed ± 20 % of the mean weight.)
Acclimatization time: 28 days
Note: In the Preliminary Experiment I, mice of 11 weeks of age (18.5-19.2 grams) and in the Preliminary Experiment II, mice of 8 weeks of age (20.7-21.2 grams) were used.

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Housing / Enrichment: Group caging / mice were provided with glass tunnel tubes
Cage type: Type II. polypropylene / polycarbonate
Bedding: Bedding was available to animals during the study
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.5 - 24.7°C
Relative humidity: 23 - 87 %
Ventilation: 15-20 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
Animals received ssniff® SM Rat/Mouse – Breeding & Maintenance, 15 mm, autoclavable Complete diet/feed for rats/mice (Batch number: 278 5652, Expiry date: 30 November 2016 and Batch number: 141 8884, Expiry date: 31 January 2017) produced by ssniff Spezialdiäten GmbH (Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany), ad libitum. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Water supply
Animals received tap water from the municipal supply from 500 mL bottles, ad libitum. Water quality control analysis was performed once every three months and microbiological assessment was performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József Attila u.36., Hungary).

Bedding
Bedding of certified wood chips especially designed to keep animals in the best natural environment was provided for animals during the study. Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + Co.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Certified nest building material was also provided for animals (ARBOCEL crinklets natural produced by J. Rettenmaier & Söhne GmbH +Co KG).

Identification and randomisation
A unique number written on the tail with a permanent marker identified each animal. The animal number was assigned on the basis of CiToxLAB Hungary Ltd.’s master file. The cages were marked with identity cards with information including study code, cage number, and dose group, sex and individual animal number. The animals were randomised and allocated to the experimental groups. The randomisation was checked by computer software using the body weight to verify the homogeneity and variability between the groups.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The formulations at 50 and 25 % (w/v) in AOO were also suitable for treatment in this study.

No. of animals per dose:

Preliminary Irritation/Toxicity Test I - 2 animals/dose
Preliminary Irritation/Toxicity Test II - 2 animals/dose
Main Experiment - 4 animals/doe

Details on study design:

Formulation
The solubility of the test item was examined in a short Preliminary Compatibility Test. The following standard OECD vehicle was assessed: AOO (acetone:olive oil 4:1 (v:v) mixture). AOO as vehicle was considered to be suitable for the study taking into account the test item characteristics, its usage and the requirements of the relevant OECD guideline. The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted). The formulations at 50 and 25 % (w/v) in AOO were also suitable for treatment in this study.
The test item was weighed and formulations prepared daily on a weight:volume basis (as % (w/v)) in the Pharmacy of CiToxLAB Hungary Ltd. Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
The Preliminary Irritation/Toxicity Test I was started according to the Study Plan on CBA/CaOlaHsd mice using two doses (2 animals/dose) at test item concentrations of 100 % (undiluted) and 50 % (w/v) in AOO. Based on the observed toxicity, an additional test was performed (Preliminary Irritation/Toxicity Test II) using two doses (2 animals/dose) at test item concentrations of 25 and 10 % (w/v) in AOO. The preliminary experiments were conducted in a similar experimental manner to the main study, but they were terminated on Day 6 and the radioactive proliferation assay was not performed.
The maximum concentration of test item in an acceptable solvent was established according to OECD guideline 429. Based on the observation of the solubility test, the maximum available concentration was 100 % (undiluted).
In the Preliminary Irritation / Toxicity Tests, all mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Both ears of each mouse were observed for erythema and scored. Ear thickness was also measured using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6. Additional quantification of the ear thickness was performed by ear punch weight determination after the euthanasia of the experimental animals.
During the Preliminary Irritation / Toxicity Test, no mortality was observed however systemic toxicity was seen: intermittent tremors were observed in the 100 % (undiluted) and 50 % (w/v) doses on Days 2-3 and hyperactivity on Day 2 in the 100 % (undiluted) and 50 % (w/v) doses and on Day 3 in the 50 % (w/v) group. Erythema (erythema score: 1) was observed for each animal in the 100 % (undiluted) and 50 % (w/v) doses on Days 2-3. Animals in the 25 and 10 % (w/v) doses were symptom-free during the test. Slightly rigid ears were observed for one animal in the 100 % (undiluted) dose group on Day 3.
No marked body weight loss ( = 5%) was detected on the mean body weight values of the groups; however one animal had slightly more than 5% body weight loss in the 50 % (w/v) dose group.
Slightly increased ear thickness values were observed in the 100 % (undiluted) dose group. The ear punch weights of all animals were within the acceptable range.
The draining auricular lymph nodes of the animals were visually examined: they were normal in the 100 % (undiluted), 25 and 10 % (w/v) groups and larger than normal in the 50 % (w/v) group (subjective judgement by analogy with observations of former experiments).
Based on these results, 100 % (undiluted) and 50 % (w/v) dose groups were considered to be too high due to the observed systemic toxicity. Therefore 25 % (w/v) dose was selected as top dose for the main test.

Topical application
During the study, animals were topically dosed with 25 µL of the appropriate formulation using a pipette on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On Day 6, animals were taken to the radioactive suite and each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (phosphate buffered saline) containing approximately 20 µCi of 3HTdR using a gauge 25G x 1" hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide (deep anaesthesia was confirmed before making incision, death was confirmed before discarding carcasses).
The draining auricular lymph nodes were excised by making a small incision on the skin between the jaw and sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. The nodes were then removed using forceps. The carcasses were discarded after cervical dislocation or after cutting through major cervical blood vessels.
Once removed, the nodes were collected in separate Petri dishes containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing. The nodes of each animal were processed individually.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs for each mouse were pelleted with a relative centrifugal force (RCF) of 190 x g (approximately) for 10 minutes at 4 °C.
After centrifugation supernatants were discarded. Pellets were gently resuspended and 10 mL of PBS was added to the tubes. The washing step was repeated twice. This procedure was repeated for each group of lymph nodes of each individual animal.

Determination of Incorporated 3HTdR
After the final washing step, supernatants were removed. Pellets were gently agitated resuspended and 3 mL of 5 % (w/v) TCA solution was added to the tubes for precipitation of macromolecules.
After overnight (approximately 18 hours) incubation at 2-8 °C, precipitates were centrifuged (approximately 190 x g for 10 minutes at 4 °C), and supernatants were removed. Pellets were resuspended in 1 mL of 5 % (w/v) TCA solution and dispersed by using an ultrasonic bath. Samples were transferred into a suitable sized scintillation vial filled with 10 mL of scintillation liquid and thoroughly mixed. The vials were loaded into a ß-scintillation counter and 3HTdR incorporation was measured (10-minute measurement).The samples were read more than once in the ß-scintillation counter to ensure accuracy and the mean result was calculated where applicable.
The ß-counter expresses the 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM). Background level was also measured in duplicates by adding 1 mL of 5 % (w/v) TCA solution into a scintillation vial filled with 10 mL of scintillation liquid.

OBSERVATIONS
Clinical Observations
During the study (Day 1 to Day 6) each animal was observed daily for any clinical signs, including local irritation and systemic toxicity. Clinical observations were performed twice a day (before and after treatments) on Days 1, 2 and 3 and once daily on Days 4, 5 and 6.
Individual records were maintained.

Measurement of Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Positive control substance(s):

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

Statistics:

DPM was measured for each animal. The average of the two measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation.
Stimulation index (SI = mean DPN of treated group divided by mean DPN of the appropriate control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

Results and discussion

Positive control results:

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. The positive control substance was examined at a concentration of 25% in the relevant vehicle (AOO) using CBA/CaOlaHsd mice.
No mortality, cutaneous reactions or signs of toxicity were observed for the positive control substance in the study. A lymphoproliferative response in line with historic positive control data (stimulation index value of 9.7) was noted for HCA in the main experiment. This value was considered to confirm the appropriate performance of the assay.
Furthermore, the DPN values observed for the vehicle and positive control substance in this experiment were within the historical control range. Each treated and control group included 4 animals.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

15.9

Results 2

Parameter:

SI

Value:

5.6

Test group / Remarks:

25 %

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

10%

Results 4

Parameter:

SI

Value:

1.3

Test group / Remarks:

5%

Cellular proliferation data / Observations:

CLINICAL OBSERVATION
No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application.

BODY WEIGHT MEASUREMENT
No treatment related effects were observed on the body weight changes of experimental animals.

PROLIFERATION ASSAY
The appearance of the lymph nodes was normal in the negative control group, in the 5 and 10 % (w/v) dose groups. Larger than normal lymph nodes were observed in the positive control group and in the 25 % (w/v) test item treated dose group.
The stimulation index values were 5.6, 1.3 and 1.3 at concentrations of 25, 10 and 5 % (w/v), respectively.

INTERPRETATION OF OBSERVATIONS
The test item was a liquid, which was formulated in AOO. Since there were no confounding effects of treatment related irritation or systemic toxicity at the applied concentrations, the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. The resulted stimulation index observed above the threshold limit of 3 at concentration of 25 % (w/v) under these exaggerated test conditions were considered to be good evidence that 3,3,5-trimethylcyclohexyl methacrylate is a sensitizer. The size of lymph nodes was in good correlation with this conclusion.
The obtained data allow the calculation of the EC3 value according to an adequate scientific method. EC3 means the effective chemical concentration required for SI=3. The calculated EC3 value of 3,3,5-trimethylcyclohexyl methacrylate is 15.9 % (w/v).

Any other information on results incl. tables

Individual Body Weights for all Animals with Group Means

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2877 2867 2868 2851	1 2 3 4	Negative (vehicle) control AOO       Mean	20.0 20.6 20.6 20.6 20.5	21.3 20.0 22.5 21.0 21.2	6.5 -2.9 9.2 1.4 3.6
2880 2855 2865 2856	5 6 7 8	3,3,5-trimethylcyclohexyl methacrylate 25% (w/v) in AOO     Mean	20.3 20.5 20.7 20.4 20.5	21.1 21.4 20.6 20.9 21.0	3.9 4.4 -0.5 2.5 2.6
2853 2871 2864 2874	9 10 11 12	3,3,5-trimethylcyclohexyl methacrylate 10% (w/v) in AOO     Mean	20.5 20.9 20.3 20.1 20.5	19.6 21.8 21.1 20.3 20.7	-4.4 4.3 3.9 1.0 1.2
2875 2857 2876 2873	13 14 15 16	3,3,5-trimethylcyclohexyl methacrylate 5% (w/v) in AOO     Mean	20.3 20.3 20.4 20.5 20.4	22.1 20.2 20.8 21.4 21.1	8.9 -0.5 2.0 4.4 3.7
2859 2879 2872 2862	17 18 19 20	Positive control 25 (w/v) % HCA in AOO     Mean	20.3 20.4 20.7 20.1 20.4	21.4 20.2 20,5 20.8 20.7	5.4 -1.0 -1.0 3.5 1.7

*: Terminal body weights were measured on Day 6.

#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

DPM, DPN and Stimulation Index Values for all Groups

Test Group Name	Identity No	Measured Total DPM (1^sttime)	Measured Total DPM (2^ndtime)	DPN (1^sttime)	DPN (2^ndtime)	Mean DPN	Group DPN	SI
Background (5% (w/v) TCA)	-	32 49	32 33	-	-	-	-	-
Negative (vehicle) control (AOO)	1 2 3 4	388 622 1006 1026	432 686 1087 1098	173.8 290.8 482.8 492.8	199.8 326.8 527.3 532.8	186.8 308.8 505.0 512.8	378.3	1.0
3,3,5-trimethylcyclohexyl methacrylate 25% (w/v) in AOO	5 6 7 8	2318 5391 5838 3504	2379 5435 5998 3606	1138.8 2675.3 2898.8 1731.8	1173.3 2701.3 2982.8 1786.8	1156.0 2688.3 2940.8 1759.3	2136.1	5.6
3,3,5-trimethylcyclohexyl methacrylate 10% (w/v) in AOO	9 10 11 12	703 1051 991 1271	753 1087 1037 1313	331.3 505.3 475.3 615.3	360.3 527.3 502.3 640.3	345.8 516.3 488.8 627.8	494.6	1.3
3,3,5-trimethylcyclohexyl methacrylate 5% (w/v) in AOO	13 14 15 16	397 306 1385 1847	414 326 1412 1890	178.3 132.8 672.3 903.3	190.8 146.8 689.8 928.8	184.5 139.8 681.0 916.0	480.3	1.3
Positive control (25% HCA in AOO)	17 18 19 20	n.d. 10140 n.d. n.d.	5974 n.d. 5513 7764	n.d. 5049.8 n.d. n.d.	2970.8 n.d. n.d. n.d.	2970.8 5049.8 2740.3 3865.8	3656.6	9.7

Notes:

1. Number of lymph nodes was 2 in case of all animals

2. n.d.: no data due to technical reasons

3. SI: Stimulation Index

Due to technical reasons occasionally no DPM result is calculated by the machine on individual samples. In these cases, all samples are rerun

and the mean result for each sample is calculated (so there is a minimum of one value for every sample).

 

 

Results of the Preliminary Irritation / Toxicity Test I

 

Individual Body Weights for all Animals with Group Means (Preliminary Experiment I)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2735 2738	1 2	100% (undiluted) 100% (undiluted) Mean	18.9 18.5 18.7	18.6 18.3 18.5	-1.6 01.1 -1.3
2739 2736	3 4	50% (w/v) 50% (w/v) Mean	18.9 19.2 19.1	18.9 18.0 18.5	0.0 -6.3 -3.1

Notes:

1. *: Terminal body weights were measure on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Experiment I)

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight^*on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
2735 2738 2739 2736	1 2 3 4	100% (undiluted) 100% (undiluted) 50% (w/v) 50% (w/v)	0.20 0.20 0.21 0.21	0.22 0.21 0.22 0.20	0.24 0.25 0.22 0.22	0.25 0.24 0.23 0.22	0.24 0.23 0.22 0.23	0.24 0.24 0.21 0.23	15.52 15.19 14.62 14.43

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

 

Summarized Clinical Observations (Preliminary Experiment I)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	100% (undiluted)	1	2735	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	2738	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	50% (w/v)	3	2739	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	2736	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 2	100% (undiluted)	1	2735	Before treatment: symptom-free, ES: 0 After treatment: hyperactivity, intermittent tremors, ES: 1
		2	2738	Before treatment: symptom-free, ES: 0 After treatment: hyperactivity, intermittent tremors, ES: 1
	50% (w/v)	3	2739	Before treatment: symptom-free, ES: 0 After treatment: hyperactivity, intermittent tremors, ES: 1
		4	2736	Before treatment: symptom-free, ES: 0 After treatment: intermittent tremors, ES: 1
DAY 3	100% (undiluted)	1	2735	Before treatment: symptom-free, ES: 0 After treatment: intermittent tremors, ES: 1
		2	2738	Before treatment: symptom-free, ES: 0 After treatment: intermittent tremors, slightly rigid ears, ES: 1
	50% (w/v)	3	2739	Before treatment: symptom-free, ES: 0 After treatment: hyperactivity, intermittent tremors, ES: 1
		4	2736	Before treatment: symptom-free, ES: 0 After treatment: intermittent tremors, ES: 1
DAY 4	100% (undiluted)	1	2735	Symptom-free, ES: 0
		2	2738	Symptom-free, ES: 0
	50% (w/v)	3	2739	Symptom-free, ES: 0
		4	2736	Symptom-free, ES: 0
DAY 5	100% (undiluted)	1	2735	Symptom-free, ES: 0
		2	2738	Symptom-free, ES: 0
	50% (w/v)	3	2739	Symptom-free, ES: 0
		4	2736	Symptom-free, ES: 0
DAY 6	100% (undiluted)	1	2735	Symptom-free, ES: 0
		2	2738	Symptom-free, ES: 0
	50% (w/v)	3	2739	Symptom-free, ES: 0
		4	2736	Symptom-free, ES: 0

Notes:

1. The clinical observation of animal on the first day was performed simultaneously with the body weight measurements

2. ES: Erythema score

 

Results of the Preliminary Irritation / Toxicity Test II

 

Individual Body Weights for all Animals with Group Means (Preliminary Experiment II)

Animal Number	Identity Number	Test Group Name	Initial Body Weight (g)	Terminal Body Weight^*(g)	Change^#(%)
2819 2803	1 2	25% (w/v) 25% (w/v) Mean	21.2 20.8 21.0	21.8 21.0 21.4	2.8 1.0 1.9
2816 2812	3 4	10% (w/v) 10% (w/v) Mean	21.2 20.7 21.0	21.5 20.0 20.8	1.4 -3.4 -1.0

Notes:

1. *: Terminal body weights were measure on Day 6

2. #: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100

 

Individual Ear Thickness for all Animals (Preliminary Experiment II)

Animal Number	Identity Number	Test Group Name	Ear Thickness on Day 1 (mm)		Ear Thickness on Day 3 (mm)		Ear Thickness on Day 6 (mm)		Biopsy weight^*on Day 6 (mg)
			Right	Left	Right	Left	Right	Left
2819 2803 2816 2812	1 2 3 4	25% (w/v) 25% (w/v) 10% (w/v) 10% (w/v)	0.21 0.20 0.21 0.20	0.21 0.21 0.20 0.20	0.25 0.23 0.24 0.23	0.26 0.23 0.23 0.23	0.22 0.24 0.23 0.22	0.24 0.22 0.23 0.23	16.71 17.46 16.27 16.37

Note:

1. *: Historical control range: 11.92-22.53 mg. Positive response is over 28.16 mg (=25%)

 

Summarized Clinical Observations (Preliminary Experiment II)

Period	Group	Animal No.	Identity No.	Clinical observations
DAY 1	25% (w/v)	1	2819	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	2803	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10% (w/v)	3	2816	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	2812	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 2	25% (w/v)	1	2819	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	2803	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10% (w/v)	3	2816	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	2812	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 3	25% (w/v)	1	2819	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		2	2803	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
	10% (w/v)	3	2816	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
		4	2812	Before treatment: symptom-free, ES: 0 After treatment: symptom-free, ES: 0
DAY 4	25% (w/v)	1	2819	Symptom-free, ES: 0
		2	2803	Symptom-free, ES: 0
	10% (w/v)	3	2816	Symptom-free, ES: 0
		4	2812	Symptom-free, ES: 0
DAY 5	25% (w/v)	1	2819	Symptom-free, ES: 0
		2	2803	Symptom-free, ES: 0
	10% (w/v)	3	2816	Symptom-free, ES: 0
		4	2812	Symptom-free, ES: 0
DAY 6	25% (w/v)	1	2819	Symptom-free, ES: 0
		2	2803	Symptom-free, ES: 0
	10% (w/v)	3	2816	Symptom-free, ES: 0
		4	2812	Symptom-free, ES: 0

Notes:

1. The clinical observation of animal on the first day was performed simultaneously with the body weight measurements

2. ES: Erythema score

 

Summarized Clinical Observations

Group	Animal No.	Identity No.	CLINICAL OBSERVATION
			DAY 1	DAY 2	DAY 3	DAY 4	DAY 5	DAY 6
Negative control (AOO)	2877   2867   2868   2851	1   2   3   4	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
3,3,5-trimethycyclohexyl methacrylate 25% (w/v) in AOO	2880   2855   2865   2856	5   6   7   8	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
3,3,5-trimethylcyclohexyl methacrylate 10% (w/v) 10% in AOO	2853   2871   2864   2874	9   10   11   12	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
3,3,5-trimethylcyclohexyl methacrylate 5% (w/v) in AOO	2875   2857   2876   2873	13   14   15   16	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free
Positive control (25% (w/v) HCA in AOO)	2859   2879   2872   2862	17   18   19   20	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free BT: symptom-free AT: symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free	Symptom-free   Symptom-free   Symptom-free   Symptom-free

Note:

1. BT: before treatment; AT: after treatment

 

Historical Control Data

Historical Control Data of the Positive and Negative Controls for CBA/CaOlaHsd mice (2014-2015)

CBA/CaOlaHsd mice
	Vehicles
	Acetone : Olive oil 4:1 (AOO)			1% Pluronic PE9200 in water (1%Plu)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	415.2	2922.6	7.5	197.7	1825.3	10.0
Range:  min                max	111.3 847.8	890.3 7674.5	3.3 15.5	23.0 680.8	154.0 6755.8	3.0 33.1
Number of cases	32	32	30	134	134	128
	Vehicles
	N,N-Dimethylformamide (DMF)			Dimethyl sulfoxide (DMSO)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	244.6	2522.6	10.8	488.7	3212.1	7.8
Range:  min                max	140.8 505.8	1201.3 4804.6	6.3 21.3	238.5 934.6	2017.2 4877.5	3.1 14.5
Number of cases	21	21	21	13	13	12
	Vehicles
	Propylene gycol (PG)			Methyl ethyl ketone (MEK)
	DPN values		SI value	DPN values		SI value
	Control	HCA 25%	HCA 25%	Control	HCA 25%	HCA 25%
Average	235.4	2371.8	10.0	260.2	4888.8	19.5
Range:  min                max	63.3 506.0	817.2 4978.0	6.5 14.4	183.5 383.3	2456.3 8682.5	8.9 36.3
Number of cases	14	14	14	9	10	10

HCA 25% = alpha-Hexylcinnamaldehyde 25% (w/v)

SI (Stimulation Index) = DPN of a treated group divided by DPN of the appropriate control group.

DPN (Disintegrations Per Node) – DPM (Disintegration Per Minute) divided by the number of lymph nodes.

In case of individual approach, SI values were calculated from the mean DPN values of the group.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In conclusion, under the conditions of the present assay, 3,3,5-trimethylcyclohexyl methacrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of 3,3,5-trimethylcyclohexyl methacrylate is 15.9 % (w/v).

Executive summary:

The aim of the study was to determine the skin sensitisation potential of 3,3,5-trimethylcyclohexyl methacrylate following dermal exposure. The study was performed with vertebrate animals as no full regulatory in vitro alternative is available. The minimum number of animals was used, corresponding to the regulatory guidelines being followed.

 

Based on the results of the Preliminary Compatibility Test, the test item characteristics, its usage and on the recommendations of the OECD Guideline no. 429, the test item was tested for formulation compatibility in acetone:olive oil 4:1 (v:v) mixture (abbreviated as AOO). The highest achievable concentration based on the regulatory requirements of the OECD guideline and the physical characteristics of the test item was 100 % (undiluted).

 

The Preliminary Irritation/Toxicity Tests were performed in CBA/CaOlaHsd mice using four doses (2 animals/dose): 100 % (undiluted), 50, 25 and 10 % (w/v) in AOO. Based on the observations recorded in the preliminary test, the 25 % (w/v) was selected as top dose for the main test.

 

In the main assay, twenty female CBA/CaOlaHsd mice were allocated to five groups of four animals each:

- three groups received 3,3,5-trimethylcyclohexyl methacrylate (formulated in AOO) at 25, 10 and 5 % (w/v) concentrations,

- the negative control group received the vehicle (AOO),

- the positive control group received 25 % (w/v) HCA (dissolved in AOO).

 

The test item solutions were applied on the dorsal surface of ears of experimental animals (25 µL/ear) for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6. At the Day 6, the cell proliferation in the local lymph nodes was measured by incorporation of tritiated methyl thymidine (3HTdR) and the values obtained were used to calculate stimulation indices (SI).

 

No mortality or signs of systemic toxicity were observed during the study. There were no indications of any irritancy at the site of application. No treatment related effects were observed on the body weight changes of experimental animals.

 

The stimulation index values were 5.6, 1.3 and 1.3 at concentrations of 25, 10 and 5 % (w/v), respectively.

 

The result of the positive control substance a-Hexylcinnamaldehyde (HCA) dissolved in the same vehicle was used to demonstrate the appropriate performance of the assay. A lymphoproliferative response in line with historic positive control data was noted for the positive control chemical, this result confirmed the validity of the assay.

 

In conclusion, under the conditions of the present assay, 3,3,5-trimethylcyclohexyl methacrylate, tested in a suitable vehicle, was shown to have sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of 3,3,5-trimethylcyclohexyl methacrylate is 15.9 % (w/v).

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS (rev. 6) 2015: Category 1 (subcategory 1B).