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Administrative data

Description of key information

Skin sensitisation: weakly sensitising. CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (LLNA EC3 value > 2%)

Respiratory sensitization: No experimental studies on respiratory sensitisation of EMA in animals have been reported in the literature, but also no clinical reports implicating EMA with the development of asthma have been published. In the absence of any positive indication, EMA is regarded as non-sensitising for the respiratory tract.

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:
24 July 2013 - 21 August 2013
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:
individual approach (adopted 22 July 2010)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(dated May 30, 2008)
GLP compliance:
yes (incl. certificate)
Remarks:
OECD Principles of GLP, as revised in 1997, ENV/MC/CHEM(98)17)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study: Pre-test: 10 - 11 weeks (beginning of treatment)
Main study: 9 - 10 weeks (beginning of treatment)
- Weight at study initiation (main experiment): 18.7 - 23.0 g (mean)
- Housing: group, Makrolon Type II (pre-test) / III (main sudy), with wire mesh top
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible
signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature 22 ±2°C
- Humidity (%): Relative humidity 45 - 94%
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test concentrations (main study): 0 (vehicle group), 25, 50, 100 % (w/v)
Test concentrations (pre-test): 50 and 100%
No. of animals per dose:
Main study: 5 females (nulliparous and non-pregnant)
Pre-test: 2 females
Details on study design:
Three groups each of five female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) once daily each on three consecutive days. A control group of five mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.

Experimental Design and Procedures
Topical Application
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1 v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ̴ 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-Methyl Thymidine
Five days after the first topical application (day 6), all mice were administered 250 µL of phosphate-buffered saline containing 81.4 µCi/mL 3HTdR (corresponds to 20.4 µCi 3HTdR per mouse) by intravenous injection via a tail vein.

Determination of Incorporated 3HTdR
Approximately five hours after treatment with 3HTdR all mice were euthanised by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.
The draining lymph nodes were rapidly excised and pooled per animal (2 nodes per animal). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a beta-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1mL-aliquots of 5 % trichloroacetic acid. The beta-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph nodes of each animal (DPM/animal) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (stimulation index, S.I.). Before DPM/animal values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than
that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical
concentrations) for either local toxicity or immunological suppression.

Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: once daily (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with 3HTdR.
Ear thickness: pre-test: prior to the first application of the test item (day1) on day 3 and before sacrifice (day 6)
Ear weights: pre-test after sacrifice; biopsy punches were taken from each ear.
Clinical signs (local / systemic): Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially
the treatment sites were observed carefully.


Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables and for the DPM values. The Dean-Dixon-Test was used for identification of possible outliers (performed with Microsoft Excel 2007).
Biological and statistical significance were considered together.


Positive control results:
Results of the GLP Positive Control

Experiment performed in April 2013.
Positive control substance: alpha-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1 v/v)
Test item conc. % (w/v) Group Measurement DPM Calculation Result
DPM-BG a) number of lymph nodes DPM per lymph node b) S.I.
--- BG I 22 --- --- --- ---
--- BG II 18 --- --- --- ---
0 1 2201 2181.0 8 272.6 1.0
5 2 3518 3498.0 8 437.3 1.6
10 3 5251 5231.0 8 653.9 2.4
25 4 12915 12895.0 8 1611.9 5.9

BG = Background (1 ml 5% trichloroacetic acid) in duplicate
1 = Control Group
2-4 = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG I and BG II
b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the
measured value by the number of lymph nodes pooled

Calculation EC3:
Test conc. % S.I.
Test Group 3 10 (a) 2.4 (b)
Test Group 4 25 (c) 5.9 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 12.6 % (w/v)
EC3 = Estimated concentration for a S.I. of 3.
a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.
Parameter:
EC3
Value:
82.6
Remarks on result:
other: Test substance: 25 % S.I.=0.93 50 % S.I.=1.41 100 % S.I.=3.85 A clear dose response was observed.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Vehicle control group (negative control with vehicle, only): 141.5 DPM per lymph node (2 lymph nodes) Test substance: 25% DPM/lymph node: 131.1 50% DPM/lymph node: 199.7 100% DPM/lymph node: 545.1

Calculation and Results of Individual Data

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

Test item concentration

DPM values measured

DPM-BG per animal
(2 lymph nodes)a)

S.I.b)

%

Group no.

Animal no.

---

---

BG I

23

---

---

---

---

BG II

24

---

---

0

1

1

149

125.5

---

0

1

2

139

115.5

---

0

1

3

217

193.5

---

0

1

4

186

162.5

---

0

1

5

134

110.5

---

25

2

6

211

187.5

1.3

25

2

7

152

128.5

0.9

25

2

8

123

99.5

0.7

25

2

9

166

142.5

1.0

25

2

10

121

97.5

0.7

50

3

11

269

245.5

1.7

50

3

12

242

218.5

1.5

50

3

13

220

196.5

1.4

50

3

14

172

148.5

1.0

50

3

15

213

189.5

1.3

100

4

16

376

352.5

2.5

100

4

17

583

559.5

4.0

100

4

18

495

471.5

3.3

100

4

19

738

714.5

5.0

100

4

20

651

627.5

4.4

1    =  Control Group

2-4=  Test Group

a)   =  values corrected for mean background value (BGI and BGII)

b)    =  Stimulation Indices relative to the mean of the control group (Group 1)

Calculation and Results of SI per Dose Group

 

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

Test item concentration % (w/v)

Group

Measurement DPM

Group Calculation

Result

mean DPM per animal (2 lymph nodes)a)

SD

 

S.I.

---

BG I

 23

---

---

---

---

BG II

 24

---

--- 

---

---

CG1

 ---

 141.5

35.5 

 1.0

25

2

 ---

 131.1

36.9

 0.93

50

3

---

 199.7

36.0

 1.41

100

4

 ---

545.1

139.8 

 3.85

 

BG  =   Background (1 ml 5% trichloroacetic acid) in duplicate

CG1=   Control Group

2-4 =   Test Group

S.I.  =   Stimulation Index

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

The EC3 value was calculated, to be 82.6% (w/v).

 

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No systemic findings were observed during the study period. On day 3 and 4 the treated animals showed an erythema of the ear skin (Score 1) and animals treated with a test concentration of 100% showed an erythema of the ear skin (Score 1) from day 3 up to day 6.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR, was within the range commonly recorded for animals of this strain and age.

The individual body weight values are included in the following table:

 

Tables of Body Weights

 

Animal No.

Dose Group

Initial Weight (g)

weight prior to treatment with3HTdR (g)

1

1

 21.3

 21.7

2

1

 22.0

 21.3

3

1

 22.3

 21.2

4

1

 20.7

 22.1

5

1

 18.8

19.8

6

2

 22.1

 23.1

7

2

 21.0

 22.1

8

2

 19.2

 20.1

9

2

 18.7

 20.6

10

2

 21.9

 20.9

11

3

 21.6

 21.6

12

3

 21.7

 23.7

13

3

 20.9

 21.6

14

3

 19.6

 21.1

15

3

 22.0

 22.8

16

4

 23.0

 22.1

 17

4

 22.3

 23.7

 18

4

 20.9

21.4

 19

 4

 21.4  21.0

 20

 4

 20.0  21.0
   Mean  21.1  21.6
   Standard Deviation

 1.2

 1.1

 

Interpretation of results:
sensitising
Conclusions:
Based on the results of a fully valid Local Lymph node assay (OECD 429, GLP), Ethyl methacrylate was considered to be a skin sensitizer.
CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (EC3 value > 2%)
Executive summary:

In a dermal sensitization study with Ethyl methacrylate (99.81%) dissolved in acetone : olive oil (4 +1 v/v) as a vehicle, 20 (5 per dose group) 9 - 10 week old female CBA/CaOlaHsd mice were tested using the method of OECD 429 (Local Lymph node Assay).  Ethyl methacrylate, three groups each of five female mice were treated daily with the test item at concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1 v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of five mice was treated with the vehicle (acetone:olive oil (4+1 v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of3H-methyl thymidine measured in a beta-scintillation counter.

The validation-/positive control experiment was performed with alpha-Hexyl cinnamic aldehyde dissolved in acetone/olive oil (4 +1 v/v). In the course of the study no cases of mortality and no signs of systemic toxicity were observed.

On day 3 and 4 the treated animals showed an erythema of the ear skin (Score 1) and animals treated with a test concentration of 100% showed an erythema of the ear skin (Score 1) from day 3 up to day 6.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 0.93, 1.41, and 3.85 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1 v/v). A clear dose response was observed. The EC3 value was calculated, to be 82.6% (w/v).

Therefore, Ethyl methacrylate was a skin sensitiser when tested in this fully valid Local Lymph Node Assay according to OECD TG 429.

CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (EC3 value > 2%)

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:

EMA is an extremely weak skin sensitiser in animals giving equivocal or negative results in the limited number of intradermal animal tests and this is consistent with a low incidence in clinical patch tests in humans. By analogy to MMA, for which a higher prevalence of contact allergy has been reported in humans and for which it has been shown has a weak potency for induction potency in LLNA studies (Betts et al., 2006), it may be concluded that EMA is a weak skin sensitiser. EMA, like other methacrylate esters, can cross-react with other methacrylates. 

In a recent mouse local lymphnode assay (LLNA) (MPA, 2013) Ethyl methacrylate formulated in acetone/olive oil (4+1 v/v) was assessed for its possible skin sensitising potential. For this purpose a LLNA assay was performed using test item concentrations of 25, 50 and 100% (w/v). All treated animals survived the scheduled study period and no signs of systemic toxicity were observed.

On day 3 and 4 the treated animals showed an erythema of the ear skin (Score 1) and animals treated with a test concentration of 100% showed an erythema of the ear skin (Score 1) from day 3 up to day 6.

In this study Stimulation Indices of 0.93, 1.41, and 3.85 were determined with the test item at concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1 v/v). A clear dose response was observed. The EC3 value was calculated, to be 82.6% (w/v).


The test item Ethyl methacrylate was found to be a skin sensitiser, the EC3 value of 82.6 % (w/v) was derived. In this study Ethyl methacrylate is a skin sensitizer of weak potency. (MPA, 2013)

--

Contact allergy to MMA is relatively common reported in the literature although closer examination of the prevalence data indicate that it is not a strong sensitizer in humans (Kimber and Pemberton, 2014). This is consistent with a weak induction potency in LLNA studies (Betts et al., 2006). Weak sensitisation potential was also demonstrated for the other members of the category in the LLNA. Preliminary data from in vitro screens confirms allergenic potential but likely due to the complexity of the ADME and reactivity processes that occur within viable, intact skin these appear not to be predictive of in vivo potency. Kimber and Pemberton concluded that “MMA and other Lower Alkyl Methacrylate esters are contact allergens, but that none of these chemicals has any more than weak skin sensitising potency.”



Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There are no clinical case studies in the literature linking exposure to EMA and the development of respiratory allergy/asthma

With the closely related methyl ester MMA, the EuRA in 2002, and later SCOEL in 2005, concluded that there have been a small number of cases reported of asthmatic reactions in people occupationally exposed to MMA in the literature but as MMA is a respiratory irritant that there is no convincing evidence that methyl methacrylate caused the acquisition of asthma in these individuals. There has been no information published since these reviews to contradict this decision. There is no evidence in the literature that exposure to MAA or the other esters within the category has caused respiratory allergy.



Justification for classification or non-classification

EMA give equivocal results in adjuvant studies in guinea pigs. In a recent LLNA assay Ethyl methacrylate was determined to be a weak skin sensitiser when tested in this fully valid Local Lymph Node Assay according to OECD TG 429 (MPA, 2013).

CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1B (EC3 value > 2%)

It is an extremely weak sensitizer in animals. No experimental studies on respiratory sensitization have been reported.