Hexyl methacrylate

Endpoint summary

Description of key information

Based on the study results, n-hexyl methacrylate is not a dermal sensitiser in the Local Lymph Node Assay. The SI obtained for n-hexyl methacrylate at all tested concentrations (highest test concentration 100% (undiluted) showed a less than threefold increase when compared to the vehicle control value, indicating no skin sensitisation potential in the LLNA assay. (MPA, 2018)

Furthermore n-hexyl methacrylate was tested in respect of its sensitizing properties in a study using two approaches:

Magnusson and Kligman's Guinea Pig Maximization Test Method  and Freund's Complete Adjuvant Test (FCAT). (Van der Walle HB, 1982)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experiment start 06 March, experiment completion April 17, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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”(adopted 22 July 2010)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Species and strain: CBA/J mice
Source: Animal Breeding Facility, Jai Research Foundation, India
Number of animals: 8 animals (2 mice/group) for preliminary assay, and 25 (5 mice/group) for main study
Sex: Female, nulliparous, non pregnant
Age of animals at starting: 9-10 weeks old at the initiation of treatment
Body weight range at starting: 19.8 – 25.3 grams
Acclimatization time: 6 days

Husbandry
Animal health: Only healthy animals were used for the study. Health status was certified by the veterinarian.
Caging : Solid floor polypropylene mice cages (size: approx. 290 mm x 220 mm x 140 mm). Each cage is fitted with a top grill having provision for keeping rodent pellet feed and water bottles. The bottom of the cages is layered with clean sterilized rice husk as the bedding material. Animals were group-housed during acclimatisation. On the days of test item application (days 0, 1 and 2), the animals were housed in individual cages. From day 3, the animals were group housed 5 mice/cage. On day 5, animals were housed in metabolic cages.
Water Bottle : Each cage was supplied with a polypropylene water bottle (capacity 300 mL) with a stainless steel nozzle.
Room Sanitation : Daily: 1. Rack was cleaned with cloth, 2. Floor of experimental procedure room was swept, 3. All work tops and the floor were mopped with a disinfectant solution.
Enrichment Material : Tunnel

Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20 - 23°C
Relative humidity: 58 - 67 %
Ventilation: minimum 15 air exchanges/hour
The temperature and relative humidity were recorded twice every day during the acclimatisation and experimental phases.

Food and feeding
- Diet: Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA, ad libitum
- Water: UV sterilised water (Reverse Osmosis water filtration system) ad libitum


Identification and randomisation
The animals were identified with appropriate labels attached to the cages indicating the study number, test item code, group number, sex, dose, cage number and animal number. Animals (except preliminary assays animals) were tattooed on paw [Animal No 1 marked with small dot on right paw (forelimb), Animal No 2 on the right and left paws (forelimb), Animal No 3 on the right and left paws (forelimb) and right paw (hindlimb), Animal No 4 on the right and left paws (forelimb and hindlimb) and Animal N° 5 had no marking] after randomisation.
After acclimatisation animals were randomised into five groups using in-house developed, validated computer software.

Vehicle:

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

Concentration:

In the preliminary assay, less than 25% ear thickness was observed at 10%, 25%, 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% concentration.
No erythema was observed at 10%, 25% 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% concentration. Therefore, dose concentrations of 25% and 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% n-hexyl methacrylate were evaluated in the main study of LLNA.

No. of animals per dose:

8 animals (2 mice/group) for preliminary assay, and 25 (5 mice/group) for main study dose

Details on study design:

Formulation
The test item was mixed with vehicle to obtain the desired dose concentrations. Fresh dose solutions were prepared prior to application on days 0, 1 and 2. Required quantity of the test item was mixed with vehicle to get the desired concentration. The concentrations of the dose solutions were not verified analytically.

ADMINISTRATION OF THE TEST ITEM
Dose Selection and Justification of Dose Selection
In the preliminary assay, less than 25% ear thickness was observed at 10%, 25%, 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% concentration.
No erythema was observed at 10%, 25% 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% concentration. Therefore, dose concentrations of 25% and 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% n-hexyl methacrylate were evaluated in the main study of LLNA.

Topical application
Three groups (G2 to G4) were treated topically for three consecutive days (days 0, 1 and 2) on the dorsal surface of both ears (25 L/ear) using a calibrated micropipette with n-hexyl methacrylate at concentrations of 10% and 50% in acetone:olive oil (4:1 v/v) and 100% n-hexyl methacrylate, respectively. Mice from vehicle control group (G1) and positive control group (G5) were handled in the same manner but received 25 L/ear of vehicle (acetone:olive oil (4:1 v/v)) and 25% a-Hexylcinnamaldehyde (v/v) in vehicle (acetone:olive oil (4:1 v/v)), respectively.

PROLIFERATION ASSAY
Injection of Tritiated Thymidine (3HTdR)
On day 5, all mice from the vehicle control, positive control and all the treatment groups were injected with 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 ± 1 µCi (740 KBq) of 3H-methyl thymidine via the tail vein.

Removal and Preparation of Draining Auricular Lymph Nodes
On day 5, 5 hours post-administration of 3H-methyl thymidine, all mice from the vehicle control, positive control and all the treatment groups were euthanised by CO2 asphyxiation. The draining auricular lymph nodes from each mouse were excised and combined in phosphate buffered saline.

Preparation of Single Cell Suspension of Lymph Node Cells
The draining auricular lymph nodes of individual mouse were collected in separate petridishes containing phosphate buffered saline (PBS). A single cell suspension of lymph node cells was prepared by gentle mechanical disaggregation through 200 to 210 µm-mesh stainless steel gauze with the plunger of the syringe and collected in a petridish. The gauze was washed with PBS into the petridish and a single cell suspension was transferred into a 15 mL graduated centrifuge tube. The single cell suspension was finally made up to 10 mL with PBS used to rinse the petridish. The cell suspension was centrifuged approximately at 190 to 200 g for 10 minutes in a centrifuge at 4 °C.
After centrifugation, the supernatant was removed by aspiration using a micropipette leaving 1 mL of supernatant above each pellet. Each pellet was gently agitated before making up to 10 mL with PBS, this procedure was repeated twice. After the final wash, the supernatant was removed leaving a minimal volume (approximate 0.5 mL) of supernatant above each pellet.
Each pellet was agitated before re-suspending with 3 mL of 5% trichloroacetic acid (TCA) and kept for precipitation of macromolecules in the refrigerator for approximately 18 hours. After incubation with 5% TCA at 4 ± 1 °C, each precipitate was recovered by centrifugation (190 to 200 g) for 10 minutes and the supernatant was removed.
The precipitate was re-suspended in 1 mL of 5% TCA. Each precipitate was transferred to a scintillation vial with 10 mL of scintillation fluid (Hionic flour) and thoroughly mixed. After minimum period of 30 minutes, the vials were loaded into a β–scintillation counter for measured the 3H-methyl thymidine incorporation. Background 3H-methyl thymidine level was measured into 1 mL aliquots of 5% TCA.

Determination of Incorporated 3HTdR
Incorporation of 3H-methyl thymidine was measured by β-scintillation counting as disintegrations per minute (DPM) for each mouse and expressed as DPM/mouse. The total radioactivity of each sample was counted using LSA (Liquid Scintillation Analyser) after 30 minutes of mixing with scintillation fluid and required quench corrections were made.
The quench curve was established using extended quench standards (PerkinElmer) of DPM β source. Computer constructed quench curve was derived from the above commercially available series of scaled standards which automatically converts Counts Per Minute (CPM) to DPM.


OBSERVATIONS
Clinical Observations
Individual animals were observed carefully for clinical signs and local irritation at the site of application and systemic toxicity. All the observations were systematically recorded for individual mice. Local irritation responses were made as per criteria given below (OECD 429, 2010; Section 22):

Observation Score
-----------------------------------------------------------------------------------------------------------------------
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beef redness) to eschar formation preventing grading of erythema) 4


Measurement of Body Weight
Body weights of individual mouse were recorded on the first day of dosing (day 0) and prior to administration of 3H-methyl thymidine (day 5). Group mean body weights were calculated.

Positive control substance(s):

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

Statistics:

In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and pair-wise comparison made between the treatment and the solvent/vehicle control group. All the parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, Student’s t-test was performed to calculate significance.

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/J 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 4.86) 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 5 animals.

Parameter:

SI

Value:

1.44

Test group / Remarks:

25 %

Parameter:

SI

Value:

2.19

Test group / Remarks:

50%

Parameter:

SI

Value:

1.68

Test group / Remarks:

100%

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. In all mice treated with 25% HCA, a local reaction consisting of erythema (score of 1) was observed from days 1 to 5 (5/5 mouse).

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

PROLIFERATION ASSAY
Proliferative responses in the draining lymph nodes were monitored by measuring the incorporation of 3H-methyl thymidine. These analyses revealed group mean DPM values of 1983.20, 2861.80, 4342.00 and 3333.20 for the vehicle control (acetone:olive oil (4:1 v/v)), 25%, 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% n-hexyl methacrylate, respectively. The DPM value for positive control (25% alpha-Hexylcinnamaldehyde) was found to be 9637.40.
A statistically significant increase in mean DPM was observed in 50% (v/v) in acetone:olive oil (4:1 v/v) and 25% (v/v) HCA when compared to vehicle control group values.

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.

A Stimulation Index (SI) of three or more (SI value of treated group over the control) indicates potential to cause skin sensitisation.
The SI obtained for n-hexyl methacrylate at all the tested concentrations showed a less than threefold increase over the control value. Therefore, n-hexyl methacrylate did not demonstrate dermal sensitisation potential in the local lymph node assay.
The SI of 4.86 obtained for the concurrent positive control, alpha-Hexylcinnamaldehyde, showed greater than a three-fold increase over the control value indicating a positive response in agreement with the historical control for this known weak sensitiser. This confirmed the reliability of this test procedure.

Skin Sensitisation Study of n-Hexyl Methacrylate by Local Lymph Node Assay in Mice

Individual Body Weights for all Animals with Group Means

	Animal Number	Test Group Name	Initial (Day 0) Body Weight (g)	Terminal ( Day 5)Body Weight^*(g)	Change^#(%)
G1	1 2 3 4 5	Negative (vehicle) control AOO       Mean	25.2 23.0 22.0 21.9 20.6 22.5	26.0 23.3 22.4 22.2 20.9 23.0	3.2 1.3 1.8 1.4 1.5 1.8
G2	6 7 8 9 10	n-hexyl methacrylate 25% (v/v) in AOO     Mean	25.3 24.4 21.7 21.3 20.4 22.6	25.7 25.0 22.0 21.8 20.6 23.0	1.6 2.5 1.4 2.3 1.0 1.8
G3	11 12 13 14 15	n-hexyl methacrylate 50% (v/v) in AOO     Mean	25.1 23.0 23.0 20.4 19.8 22.3	25.7 23.5 23.3 20.6 20.1 22.6	2.4 2.2 1.3 1.0 1.5 1.7
G4	16 17 18 19 20	n-hexyl methacrylate 100% (undiluted)     Mean	24.1 23.0 22.0 21.8 20.3 22.2	24.6 23.2 23.0 22.5 21.0 22.9	2.1 0.9 4.5 3.2 3.4 2.8
G5	21 22 23 24 25	Positive control 25 (v/v) % HCA in AOO     Mean	25.1 23.0 22.4 20.9 20.6 22.4	25.3 24.0 22,6 21.6 21.1 22.9	0.8 4.3 0.9 3.3 2.1 2.3

*: Terminal body weights were measured on Day 5.

#: = (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	DPM*	Group DPM	Standard Deviation	SI
G1 Negative (0% vehicle) control (AOO)	1 2 3 4 5	2173 1860 2036 855 3062	2159 1846 2022 841 3048	1983.20	788.93	1.0
G2 n-hexyl methacrylate 25% (v/v) in AOO	6 7 8 9 10	3771 2740 2554 1592 3722	3757 2726 2540 1578 3708	2861.80	906.58	1.44
G3 n-hexyl methacrylate 50% (v/v) in AOO	11 12 13 14 15	2325 5574 6052 4502 3327	2311 5560 6038 4488 3313	4342.00**	1546.37	2.19
G4 n.-hexyl methacrylate 100% (undiluted)	16 17 18 19 20	3896 2736 3628 4350 2126	3882 2722 3614 4336 2112	3333.20	901.46	1.68
G5 Positive control (25% (v/v) HCA in AOO)	21 22 23 24 25	17387 6317 8270 10550 5733	17373 6303 8256 10536 5719	9637.40 **	4717.71	4.86

Notes: DPM = Disintegration per minute, Measured Background DPM of 5% TCA = 14,

Measured DPM= Value measured by Liquid Scintillation Analyser,

DPM* = Measured DPM (individual animal) - Background DPM,

HCA = α-Hexylcinnamaldehyde,Vehicle = Acetone : Olive oil (4:1 v/v)

Stimulation Index= mean DPM of test group divided by mean DPM of solvent/vehicle control group

** = significantly higher than control (p ≤0.05)

 

                                                                                                                                                                                                                                                                                                                                                                                                Sex: Dermal irritation Scores              Sex: Female

Group N°	Dose Concentration (%)	Mouse N°	Erythema scores on Day
			0	1	2	3	4	5
G1	0% (Vehicle control)	1	0	0	0	0	0	0
		2	0	0	0	0	0	0
		3	0	0	0	0	0	0
		4	0	0	0	0	0	0
		5	0	0	0	0	0	0
G2	25% (v/v)n-Hexyl Methacrylate	6	0	0	0	0	0	0
		7	0	0	0	0	0	0
		8	0	0	0	0	0	0
		9	0	0	0	0	0	0
		10	0	0	0	0	0	0
G3	50% (v/v)n-Hexyl Methacrylate	11	0	0	0	0	0	0
		12	0	0	0	0	0	0
		13	0	0	0	0	0	0
		14	0	0	0	0	0	0
		15	0	0	0	0	0	0
G4	100%n-Hexyl Methacrylate(undiluted)	16	0	0	0	0	0	0
		17	0	0	0	0	0	0
		18	0	0	0	0	0	0
		19	0	0	0	0	0	0
		20	0	0	0	0	0	0
G5	25% (v/v) HCA	21	0	1	1	1	1	1
		22	0	1	1	1	1	1
		23	0	1	1	1	1	1
		24	0	1	1	1	1	1
		25	0	1	1	1	1	1

Key:  0 = No erythema, 1 = Very slight erythema (barely perceptible), HCA = α-Hexylcinnamaldehyde, Vehicle = Acetone:Olive oil (4:1 v/v)

Clinical Observations of Individual Mouse other than Irritation Response

 Sex: Female

Group N°	Dose Concentration (%)	Mouse N°	Clinical Observation on Day
			0	1	2	3	4	5
G1	0% (Vehicle control)	1	1	1	1	1	1	1
		2	1	1	1	1	1	1
		3	1	1	1	1	1	1
		4	1	1	1	1	1	1
		5	1	1	1	1	1	1
G2	25% (v/v)n-Hexyl Methacrylate	6	1	1	1	1	1	1
		7	1	1	1	1	1	1
		8	1	1	1	1	1	1
		9	1	1	1	1	1	1
		10	1	1	1	1	1	1
G3	50% (v/v)n-Hexyl Methacrylate	11	1	1	1	1	1	1
		12	1	1	1	1	1	1
		13	1	1	1	1	1	1
		14	1	1	1	1	1	1
		15	1	1	1	1	1	1
G4	100%n-Hexyl Methacrylate(undiluted)	16	1	1	1	1	1	1
		17	1	1	1	1	1	1
		18	1	1	1	1	1	1
		19	1	1	1	1	1	1
		20	1	1	1	1	1	1
G5	25% (v/v) HCA	21	1	1	1	1	1	1
		22	1	1	1	1	1	1
		23	1	1	1	1	1	1
		24	1	1	1	1	1	1
		25	1	1	1	1	1	1

Key: HCA = α-Hexylcinnamaldehyde, Vehicle = Acetone:Olive oil (4:1 v/v)

Clinical Sign: 1 = Normal

Results of Preliminary Assay

 

Dermal Irritation Scores                                                                                          Sex: Female

Group N°	Dose Concentration (%)	Mouse N°	Erythema on Days
			0	1	2	3	4	5
G1	10% (v/v) n-Hexyl Methacrylate	1	0	0	0	0	0	0
		2	0	0	0	0	0	0
G2	25% (v/v) n-Hexyl Methacrylate	3	0	0	0	0	0	0
		4	0	0	0	0	0	0
G3	50% (v/v) n-Hexyl Methacrylate	5	0	0	0	0	0	0
		6	0	0	0	0	0	0
G4	100% n-Hexyl Methacrylate (undiluted)	7	0	0	0	0	0	0
		8	0	0	0	0	0	0

Note: 0 = No erythema, Vehicle = Acetone:Olive oil (4:1 v/v)

Group Mean Body Weight

Group N°	Dose Concentration (%)	N° of Mice Used	Mean Body Weight (g)
			Day 0	Day 5
G1	10% (v/v) n-Hexyl Methacrylate	2	22.6 ± 3.1	23.4 ± 3.3
G2	25% (v/v) n-Hexyl Methacrylate	2	21.3 ± 0.6	22.2 ± 1.0
G3	50% (v/v) n-Hexyl Methacrylate	2	20.7 ± 2.8	21.3 ± 2.3
G4	100% n-Hexyl Methacrylate (undiluted)	2	19.9 ± 1.6	21.5 ± 2.3

Note: Vehicle = Acetone : Olive oil (4:1 v/v)

 

Summary of Ear Thickness

Group N°	Dose Concentration (%)	Ear Thickness (mm) on Day (Mean± SD)
		0		2		5
		Left	Right	Left	Right	Left	Right
G1	10% (v/v) n-Hexyl Methacrylate	0.251 ± 0.002	0.251 ± 0.001	0.264 ± 0.002	0.264 ± 0.004	0.268 ± 0.004	0.265 ± 0.001
G2	25% (v/v) n-Hexyl Methacrylate	0.240 ± 0.016	0.233 ± 0.015	0.260 ± 0.016	0.255 ± 0.015	0.267 ± 0.014	0.262 ± 0.013
G3	50% (v/v) n-Hexyl Methacrylate	0.253 ± 0.023	0.246 ± 0.021	0.278 ± 0.018	0.272 ± 0.018	0.292 ± 0.025	0.286 ± 0.024
G4	100% n-Hexyl Methacrylate(undiluted)	0.231 ± 0.004	0.234 ± 0.004	0.274 ± 0.008	0.277 ± 0.002	0.282 ± 0.008	0.284 ± 0.006

Summary of Ear Thickness Percent Change

Group N°	Dose Concentration (%)	N° of Mice Used	Mean Ear Thickness (percent change)(Mean± SD)
			Left Ear Thickness (% Change)		Right Ear Thickness (% Change)
			Day 2	Day 5	Day 2	Day 5
G1	10% (v/v) n-Hexyl Methacrylate	2	5.190 ± 0.044	6.983 ± 0.788	5.186 ± 2.283	5.578 ± 0.031
G2	25% (v/v) n-Hexyl Methacrylate	2	8.590 ± 0.879	11.539 ± 1.669	9.482 ± 0.606	12.519 ± 1.408
G3	50% (v/v) n-Hexyl Methacrylate	2	9.996 ± 2.571	15.241 ± 0.525	10.868 ± 1.772	16.494 ± 0.062
G4	100% n-Hexyl Methacrylate(undiluted)	2	18.388 ± 1.193	22.065 ± 1.431	18.423 ± 0.885	21.407 ± 0.887

Note: Vehicle = Acetone : Olive oil (4:1 v/v)

Individual Animal Ear Thickness Measurement (mm)

Group N°	Dose Concentration (%)	Mouse N°	Day 0		Day 2		Day 5
			Left	Right	Left	Right	Left	Right
G1	10% (v/v) n-Hexyl Methacrylate	1	0.252	0.250	0.265	0.267	0.271	0.264
		2	0.249	0.252	0.262	0.261	0.265	0.266
G2	25% (v/v) n-Hexyl Methacrylate	3	0.251	0.243	0.271	0.265	0.277	0.271
		4	0.228	0.222	0.249	0.244	0.257	0.252
G3	50% (v/v) n-Hexyl Methacrylate	5	0.237	0.231	0.265	0.259	0.274	0.269
		6	0.269	0.260	0.291	0.285	0.309	0.303
G4	100% n-Hexyl Methacrylate(undiluted)	7	0.228	0.231	0.268	0.275	0.276	0.279
		8	0.234	0.236	0.279	0.278	0.288	0.288

Note: Vehicle = Acetone : Olive oil (4:1 v/v)

 

Clinical Observations of Individual Mouse other than Irritation Response

Group N°	Dose Concentration (%)	Mouse N°	Clinical Observation on Day
			0	1	2	3	4	5
G1	10% (v/v) n-Hexyl Methacrylate	1	1	1	1	1	1	1
		2	1	1	1	1	1	1
G2	25% (v/v) n-Hexyl Methacrylate	3	1	1	1	1	1	1
		4	1	1	1	1	1	1
G3	50% (v/v) n-Hexyl Methacrylate	5	1	1	1	1	1	1
		6	1	1	1	1	1	1
G4	100% n-Hexyl Methacrylate(undiluted)	7	1	1	1	1	1	1
		8	1	1	1	1	1	1

Clinical Sign: 1 = Normal

Key: Vehicle = Acetone:Olive oil (4:1 v/v)

Interpretation of results:

GHS criteria not met

Remarks:

Not a skin sensitiser.

Conclusions:

In conclusion, under the conditions of the present assay, n-Hexyl methacrylate, tested in a suitable vehicle, indicating no skin sensitisation potential (not a sensitizer) in the Local Lymph Node Assay. Based on this study result, n-hexyl methacrylate is not a dermal sensitiser.
Based on the results of this study, an indication of the classification for n-hexyl methacrylate is as follows:
Globally Harmonized System of Classification and Labeling of Chemicals (GHS 2017): Not classified as skin sensitiser

Executive summary:

The aim of the study was to determine the skin sensitisation potential of n-hexyl methacrylate following dermal exposure.

 

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/J mice using three doses (2 animals/dose): 100 % (undiluted), 50 and 25 % (v/v) in AOO. Based on the observations recorded in the preliminary test, the 100 % (v/v) was selected as top dose for the main test.

 

In the main assay, twentyfive female CBA/J mice were allocated to five groups of five animals each:

- three groups received n-Hexyl methacrylate (formulated in AOO) at 100, 50, and 25 % (v/v) concentrations,

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

- the positive control group received 25 % (v/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 0, 1 and 2). There was no treatment on Days 3and 4. At the Day 5, 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 1.68, 2.19 and 1.44 at concentrations of 100, 50 and 25 % (v/v), respectively.

 

The result of the positive control substance alpha-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, n-hexyl methacrylate, tested in a suitable vehicle, was shown to have no sensitisation potential (not a sensitizer) in the Local Lymph Node Assay.The SI obtained for n-hexyl methacrylate at all tested concentrations showed a less than threefold increase when compared to the vehicle control value, indicating no skin sensitisation potential in the LLNA assay.

 

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS 2017: Not classified as skin sensitiser.

NOTE: Any of data in this dataset are disseminated by the European Union on a right-to-know basis and this is not a publication in the same sense as a book or an article in a journal. The right of ownership in any part of this information is reserved by the data owner(s). The use of this information for any other, e.g. commercial purpose is strictly reserved to the data owners and those persons or legal entities having paid the respective access fee for the intended purpose.

Reference 2

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assesment

Remarks:

DATA QUALITY: Study was conducted in accordance with recognized scientific standards for analyzing delayed contact sensitivity. Although no positive control indicated, the authors of this study are referenced experts in the field of contact dermal sensitization and this factor alone does not detract from the scientific reliability of the study.

Qualifier:

according to guideline

Guideline:

other: see publication or details on study design

Principles of method if other than guideline:

Method: Freund`s complete adjuvant test (FCAT)

GLP compliance:

not specified

Type of study:

Freund's complete adjuvant test

Specific details on test material used for the study:

Purity 97 %

Species:

guinea pig

Strain:

other: two strains were used in this test battery with 2 approaches and 14 substances

Sex:

female

Details on test animals and environmental conditions:

Female albino guinea pigs of the Dunkin-Hartley strain and Himalayan white spotted,  weighing 350-450 grams. Animals were housed in 
pairs and fed a diet  supplemented with Vitamin C.

Route:

intradermal

Vehicle:

other: mixture of 2 parts methyl ethyl ketone and 1 part peanut oil (Aramek) per volume

Concentration / amount:

0.025 mL

Route:

epicutaneous, occlusive

Vehicle:

other: mixture of 2 parts methyl ethyl ketone and 1 part peanut oil (Aramek) per volume

Concentration / amount:

0.025 mL

No. of animals per dose:

GMPT: 10 g. pigs per experimental group and 6 g.  pigs in control.
FCAT: 8 animals in the experimental group and 4-6 in  the control.

Details on study design:

The maximum non-irritating  concentration was determined. Skin irritation caused by a single open  application 
was determined in FCA pretreated animals; reactions were read  at 24 and 48 hours after application. In the 
GMPT 0.5 M  used on day 0  and 1 M on day 7; in the FCAT 5 x 0.5 M used on induction days 0-9.

Magnusson and Kligman's Guinea Pig Maximization Test Method  and Freund's Complete Adjuvant Test (FCAT). 
All animals were clipped free  of hair in the shoulder region of application.

GPMT:        
 Induction Phase A: Day 0, 3 pairs of intradermal injections of Freund's  Complete Adjuvant (FCA) mixed with 
test substance or vehicle control  performed in the interscapular region. Three injections of 0.1 ml  
administered as follows: 0.1 ml of adjuvant (FCA) alone; 0.1 ml of test  substance in Aramek (peanut oil); 
and 0.1 ml of test substance in  adjuvant.
       
Induction Phase B: Same area was shaved with electric razor. Test  material was dissolved in ethanol (80%) 
in a concentration giving a  slight to moderated irritation was applied. The occlusive wrap was  removed 
after 48 hours of contact.
        
Challenge Phase: Day 21. Occluded topical application of a maximum  non-irritating dose. The left flank 
served as control and received  vehicle only.  Test substance was applied. Occlusive bandage and test  
material were applied and held in contact for 24 hours.        

Scoring: Skin reactions were evaluated at 24 and 48 hrs after removal of  dressing.        

Challenge Phase: Day 35. Left flank was shaved. Control and  experimental groups were again treated 
as before (test  material in  vehicle or vehicle only);  test site was non-occluded; and test sites  read 
24 and 48 hours later.        

Positive Control: None used    

FCAT:        
 Induction Phase: Test material was emulsified in adjuvant (FCA) and  distilled water.  On days 0, 2, 4, 7 
and 9 intradermal injections of 0.1  ml were given in the shoulder region, across the back.         
Challenge Phase: All animals were test epicutaneously on day 21 (right  flank) and day 35 (left flank). 
Test site was non-occluded; and test  sites read 24 and 48 hours later.                 
Scoring: Skin reactions were evaluated at 24 and 48 hrs after removal of  dressing.

Challenge controls:

exposed to the vehicle only in the same manner as experimental animals

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

3 M

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

GPMT

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5 * 0.5 M

No. with + reactions:

3

Total no. in group:

8

Clinical observations:

strong redness plus swelling

Remarks on result:

positive indication of skin sensitisation

Remarks:

FCAT

To determine the sensitization potential to guinea pigs by two different test methods.

 

RESULTS/OBSERVATIONS:

GMPT: 0/10 guinea pigs responded on challenge days 21 and 35.

FCAT: 3/8 g. pigs responded on days 21 and 35 and the test material was classified as a Grade II-3 (meaning the intensity of the sensitizing reaction was "3- strong redness plus swelling").

Interpretation of results:

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

Conclusions:

Classification: sensitizing 1B

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation study in mice (MPA, 2018):

The aim of the study was to determine the skin sensitisation potential of n-hexyl methacrylate following dermal exposure. The study was performed with vertebrate animals.

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

 

In the main assay, twentyfive female CBA/J mice were allocated to five groups of five animals each:

- three groups received n-Hexyl methacrylate (formulated in AOO) at 100, 50, and 25 % (v/v) concentrations,

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

- the positive control group received 25 % (v/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 0, 1 and 2). There was no treatment on Days 3and 4. At the Day 5, 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 1.68, 2.19 and 1.44 at concentrations of 100, 50 and 25 % (v/v), respectively.

 

The result of the positive control substance alpha-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, n-hexyl methacrylate, tested in a suitable vehicle, was shown to have no sensitisation potential (not a sensitizer) in the Local Lymph Node Assay.The SI obtained for n-hexyl methacrylate at all tested concentrations showed a less than threefold increase when compared to the vehicle control value, indicating no skin sensitisation potential in the LLNA assay.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Short description of key information:

No cases of respiratory allergy have been reported in the literature.

Inhalation exposure is not considered as a relevant pathway of exposure for n-Hexyl methacrylate.

Therefore, respiratory sensitisation has not to be addressed.

Justification for classification or non-classification

The studies conducted with n-HMA showed contradictory results, in the GPMT and LLNA assay (MPA, 2018) n-HMA was not sensitizing in guinea pigs and CBA/J strain mice but the FCAT revealed sensitization in three out of eight animals.

Based on the findings of the FCAT part in this study n-HMA has skin sensitizing properties and is classified as follows.

According to EU-GHS (CLP): Hazard subcategory: 1B H317

According to UN-GHS (CLP): Hazard subcategory: 1B H317