Registration Dossier

Administrative data

Description of key information

n-octyl methacrylate was shown to have weak sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of n-octyl methacrylate is 31.82 % (v/v). (MPA, 2018)

A further study for the endpoint of skin sensitization with questionable validity is published with n-octyl methacrylate (Kanazawa et al. Contact Dermatitis 40(1): 19 - 23 (1999)).

The weak sensitisation potential of n-octyl methacrylate is supported by the read across using the category approach lower alkyl (C1-C8) methacrylates which was used to predict the sensitizising properties of n-octyl methacrylate. As source substances 2-ethylhexyl methacrylate was chosen.

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:
Experiment start 06 March, experiment completion April 11, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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. certificate)
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: 10-11 weeks old at the initiation of treatment
Body weight range at starting: 20.3 – 25.6 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:
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:
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-oct
yl 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 µL/ear) using a calibrated micropipette with n-octyl methacrylate at concentrations of 25% and 50% in acetone:olive oil (4:1 v/v) and 100% (undiluted) n-octyl methacrylate, respectively. Mice from vehicle control group (G1) and positive control group (G5) were handled in the same manner but received 25 µL/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 measuring 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 5.83) 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:
2.55
Test group / Remarks:
25 %
Parameter:
SI
Value:
4.2
Test group / Remarks:
50%
Parameter:
SI
Value:
4.98
Test group / Remarks:
100%
Key result
Parameter:
EC3
Remarks:
%
Value:
31.82
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 2 to 4 (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 100% n-octyl methacrylate 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-octyl methacrylate at 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% concentration showed a greater than threefold increase compared to the vehicle control value with an EC3 value found to be 31.82%. Therefore, n-octyl methacrylate demonstrates weak dermal sensitisation potential in the local lymph node assay.
The SI of 5.83 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-Octyl 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

25.6

23.0

22.3

21.5

20.8

22.6

26.3

24.0

23.0

22.2

22.0

23.5

2.7

4.3

3.1

3.3

5.8

3.8

G2

6

7

8

9

10

n-octyl methacrylate 25% (v/v) in AOO

 

 

Mean

25.6

23.5

22.7

21.9

20.6

22.9

26.3

24.0

23.4

22.1

21.0

23.4

2.7

2.1

3.1

0.9

1.9

2.1

G3

11

12

13

14

15

n-octyl methacrylate 50% (v/v) in AOO

 

 

Mean

25.0

23.4

22.4

21.0

20.3

22.4

25.3

23.9

23.0

21.5

20.8

22.9

1.2

2.1

2.7

2.4

2.5

2.2

G4

16

17

18

19

20

n-octyl methacrylate 100% (undiluted)

 

 

Mean

25.4

23.0

22.3

22.0

21.0

22.7

26.2

24.0

22.8

22.7

22.0

23.5

3.1

4.3

2.2

3.2

4.8

3.5

G5

21

22

23

24

25

Positive control 25 (v/v) % HCA in AOO

 

 

Mean

23.9

23.0

22.0

21.5

20.9

22.3

24.5

23.6

23.0

22.1

21.5

22.9

2.5

2.6

4.5

2.8

2.9

3.1

*: 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

1218

644

986

1425

879

1169

595

937

1376

830

 

981.40

302.12

1.0

G2

n-octyl methacrylate 25% (v/v) in AOO

6

7

8

9

10

2293

2187

3419

2500

2378

2244

2138

3370

2451

2329

2506.40

496.21

2.55

G3

n-octyl methacrylate 50% (v/v) in AOO

11

12

13

14

15

5397

2705

3228

4921

4583

5348

2656

3179

4872

4534

4117.80*

1148.22

4.20

G4

n.-octyl methacrylate 100% (undiluted)

16

17

18

19

20

4812

3884

4634

7091

4264

7463

3835

4585

7042

4215

4888.00*

1255.87

4.98

G5

Positive control (25% (v/v) HCA in AOO)

21

22

23

24

25

4780

6927

5833

6687

4629

4731

6878

5784

6638

4580

5722.20*

1056.58

5.83

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 (p0.01)

 

                                                                                                                                                                                                                                                                                                                                                                                                Sex: Dermal irritation Scores              Sex: Female

Group N°

Dose Concentration (%)

Mouse

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-Octyl 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-Octyl 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-Octyl 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

0

1

1

1

0

22

0

0

1

1

1

0

23

0

0

1

1

1

0

24

0

0

1

1

1

0

25

0

0

1

1

1

0

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-Octyl 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-Octyl 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-Octyl 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

Dose

Concentration (%)

Mouse N°

Erythema on Days

0

1

2

3

4

5

G1

10% (v/v) n-Octyl Methacrylate

1

0

0

0

0

0

0

2

0

0

0

0

0

0

G2

25% (v/v) n-Octyl Methacrylate

3

0

0

0

0

0

0

4

0

0

0

0

0

0

G3

50% (v/v) n-Octyl Methacrylate

5

0

0

0

0

0

0

6

0

0

0

0

0

0

G4

100% n-Octyl 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-Octyl Methacrylate

2

22.2 ± 0.4

22.5 ± 1.0

G2

25% (v/v) n-Octyl Methacrylate

2

24.1 ± 1.8

24.8 ± 1.6

G3

50% (v/v) n-Octyl Methacrylate

2

22.2 ± 0.4

22.6 ± 0.3

G4

100% n-Octyl Methacrylate (undiluted)

2

24.0 ± 1.0

25.0 ± 0.6

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-Octyl Methacrylate

0.240

±

0.016

0.236

±

0.018

0.248

±

0.013

0.243

±

0.018

0.256

±

0.017

0.250

±

0.020

G2

25% (v/v) n-Octyl Methacrylate

0.243

±

0.001

0.237

±

0.004

0.254

±

0.000

0.250

±

0.003

0.264

±

0.002

0.259

±

0.001

G3

50% (v/v) n-Octyl Methacrylate

0.252

±

0.004

0.239

±

0.010

0.276

±

0.0002

0.267

±

0.008

0.286

±

0.003

0.275

±

0.010

G4

100% n-Octyl Methacrylate(undiluted)

0.252

±

0.005

0.245

±

0.007

0.294

±

0.007

0.287

±

0.008

0.307

±

0.007

0.303

±

0.010

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-Octyl Methacrylate

2

3.379

±

1.398

6.662

±

0.158

2.775

±

0.515

5.927

±

0.137

G2

25% (v/v) n-Octyl Methacrylate

2

4.743

±

0.305

8.662

±

1.191

5.492

±

0.695

9.087

±

1.654

G3

50% (v/v) n-Octyl Methacrylate

2

9.334

±

0.999

13.449

±

0.788

11.738

±

1.078

15.076

±

0.624

G4

100% n-Octyl Methacrylate (undiluted)

2

16.894

±

0.511

22.064

±

0.409

17.142

±

0.083

23.667

±

0.472

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-Octyl Methacrylate

1

0.229

0.223

0.239

0.230

0.244

0.236

2

0.251

0.249

0.257

0.255

0.268

0.264

G2

25% (v/v) n-Octyl Methacrylate

3

0.243

0.240

0.254

0.252

0.262

0.259

4

0.242

0.234

0.254

0.248

0.265

0.258

G3

50% (v/v) n-Octyl Methacrylate

5

0.255

0.246

0.277

0.273

0.288

0.282

6

0.249

0.232

0.274

0.261

0.284

0.268

G4

100% n-Octyl Methacrylate(undiluted)

7

0.255

0.250

0.299

0.293

0.312

0.310

8

0.248

0.240

0.289

0.281

0.302

0.296

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-Octyl Methacrylate

1

1

1

1

1

1

1

2

1

1

1

1

1

1

G2

25% (v/v) n-Octyl Methacrylate

3

1

1

1

1

1

1

4

1

1

1

1

1

1

G3

50% (v/v) n-Octyl Methacrylate

5

1

1

1

1

1

1

6

1

1

1

1

1

1

G4

100% n-Octyl 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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
In conclusion, under the conditions of the present assay, n-Octyl methacrylate, tested in a suitable vehicle, indicating weak skin sensitisation potential in the Local Lymph Node Assay. Based on this study result, n-octyl methacrylate is a dermal sensitiser.
Based on the results of this study, an indication of the classification for n-octyl methacrylate is as follows:
Globally Harmonized System of Classification and Labeling of Chemicals (GHS 2017): Classified as a weak skin sensitiser, Cat 1B
Executive summary:

The aim of the study was to determine the skin sensitisation potential of n-octyl 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 % (w/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-octyl 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 3 and 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 2.55, 4.20 and 4.98 at concentrations of 100, 50 and 25 % (w/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-octyl methacrylate, tested in a suitable vehicle, was shown to have weak sensitisation potential in the Local Lymph Node Assay. The SI obtained for n-octyl methacrylate at 50% (v/v) in acetone:olive oil (4:1 v/v) and 100% n-octyl methacrylate concentrations showed a greater than threefold increase compared to the vehicle control value with an EC3 value found to be 31.82% (v/v) in the LLNA assay.

The following classification/labelling is triggered:

Regulation (EC) No 1272/2008 (CLP) / GHS 2017: Classified as skin sensitiser, Cat.: 1B

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

n-Octyl methacrylate:

Skin sensitisation study in mice (MPA, 2018):

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

The Preliminary Irritation/Toxicity Tests were performed in CBA/J mice using four doses (2 animals/dose): 100 % (undiluted), 50, 25 and 10 % (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-octyl methacrylate (formulated in AOO) at 25, 50 and 100 % (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 3 and 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 2.55, 4.20 and 4.98 at concentrations of 25, 50 and 100 % (v/v), respectively.

In conclusion, under the conditions of the present assay, n-octyl methacrylate, tested in a suitable vehicle, was shown to have weak sensitisation potential (sensitizer) in the Local Lymph Node Assay. The calculated EC3 value of n-octyl methacrylate is 31.82 % (v/v).

Read across to 2 -Ethylhexyl methacrylate:

Animal data

The sensitizing potential of 2-ethylhexyl methacrylate was evaluated in three independent studies performed by intradermal injection and topical applications on guinea pigs according to a modified Magnusson and Kligman method.

In the's study (1981), the induction phase has been realized both by intradermal route on day 1 (5 % in paraffin) and by cutaneous route on day 6 -7 (undiluted) in 2 groups of guinea pigs: 5 females for control group and 10 females for treated group. The challenge phase was realized on day 21 by cutaneous application of 3 % on petrolatum on the flank; the cutaneous reactions were scored 48 and 72 hours after the challenge phase. Either mild or well-defined erythema was observed in three of 10 animals 48 hours following challenge applications. This effect was accompanied by slight oedema in two animals at 72 hours. This represents a 30% sensitization rate; therefore, the product is considered to be a Moderate Contact Allergen according to the Maximization grading system.

In another guideline GPMT study where 2-EHMA (Allen, 1999) was tested at an intradermal induction concentration of 1 % in water (w/v) and an epicutaneus induction concentration of 100 %, 0/20 animals reacted when challenged with 75 % or undiluted 2 -EHMA after 24 or 48 h after the challenge application. 2 -EHMA was non-sensitising in this test.

In the Clemmensen study (1984), the induction phase has been realized both by intradermal route on day 1 (5 or 25 %) and by cutaneous route on day 7 -8 (undiluted) in 2 groups of 20 or 12 female guinea pigs, respectively. The challenge phase was realized on day 21 by cutaneous application of 3 or 25% solution; the cutaneous reactions were scored 48 and 72 hours after the challenge phase. No positive skin reaction was observed in the 20 animals challenged with 3% and positive skin reaction was observed in two of 12 animals challenged with 25%. This represents a 16% sensitization rate; therefore, the product is considered to be a Mild Contact Allergen according to the Maximization grading system.

In a fully valid LLNA according to OECD 429, treatment with 2-EHMA caused Stimulation Indices (S.I.) of 1.53, 2.66, and 2.85 at concentrations of 25, 50, and 100% in acetone:olive oil (4+1). A clear dose response was observed, however the treshold of 3 was not reached indicating no skin sensitizing potential of the test item in this test system relevant for classification. (MPA, 2013)

Human data

There are very limited patch test data on 2-EHMA which do not allow drawing a conclusion regarding prevalence or potency. By analogy to the other lower alkyl methacrylates it is expected that 2 -EHMA is a skin sensitiser of low potency in humans.


Short description of key information:

n-Octyl methacrylate is considered as weak skin sensitizer in a LLNA. Calculated EC3 value of n-octyl methacrylate was 31.82 % (v/v). (MPA, 2018)

Read across to 2-ethylhexyl methacrylate structural analogue substance and category member:

2-ethylhexyl methacrylate was a mild to moderate skin sensitizer in guinea pig maximization tests.

2-ethylhexyl methacrylate is not considered as skin sensitizer in a fully valid LLNA. (MPA, 2013)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Short description of key information:
Inhalation exposure is not considered as a relevant pathway of exposure for 2-EHMA and n-Octyl methacrylate.

Therefore, respiratory sensitisation has not to be addressed.

Justification for classification or non-classification

Based on the experimental data, n-octyl methacrylate is considered as skin sensitiser of low potency and has therefore to be classified as skin sensitizing, category 1, subcategory 1B, according to the Regulation EC n°1272/2008.

Justification : EC3 value of 31.82 % in LLNA study.

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

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