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Administrative data

Description of key information

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 - 14 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study OECD 429, GLP.
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): 18.8 - 23.7 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. The Dean-Dixon-Test and the Grubb's test were 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)
Test item concentration % (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.
Key result
Parameter:
EC3
Value:
43.6
Remarks on result:
other: Test substance: 25 % S.I.=2.19 50 % S.I.=3.28 100 % S.I.=5.41 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: 309.3 50% DPM/lymph node: 464.1 100% DPM/lymph node: 764.9

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

281

257.5

1.8

25

2

7

268

244.5

1.7

25

2

8

467

443.5

3.1

25

2

9

329

305.5

2.2

25

2

10

319

295.5

2.1

50

3

11

376

352.5

2.5

50

3

12

698

674.5

4.8

50

3

13

596

572.5

4.0

50

3

14

398

374.5

2.6

50

3

15

370

346.5

2.4

100

4

16

743

719.5

5.1

100

4

17

531

507.5

3.6

100

4

18

840

816.5

5.8

100

4

19

1164

1140.5

8.1

100

4

20

664

640.5

4.5

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)

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

 ---

 309.3

79.2

 2.19

50

3

---

 464.1

150.3

 3.28

100

4

 ---

764.9

238.5 

 5.41

 

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 43.6% (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, all test item treated animals showed an erythema of the ear skin (Score 1). On days 4 and 5 the animals treated with 25 and 50% of n-Butyl methacrylate 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 2). Furthermore, on day 6 the animals treated with a test item concentration of 50 and 100% showed an ear skin erythema Score 1.

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

 21.4

 21.8

7

2

 20.4

 20.4

8

2

 23.6

 23.1

9

2

 20.7

 22.3

10

2

 22.5

 24.0

11

3

 22.8

 22.8

12

3

 23.7

 24.0

13

3

 20.8

 22.1

14

3

 20.5

 21.5

15

3

 21.1

 21.7

16

4

 20.7

 23.4

 17

4

 23.2

 23.9

 18

4

 20.9

20.5

 19

 4

 21.0  21.8

 20

 4

 18.9  20.0
   Mean  21.4  22.0
   Standard Deviation

 1.4

 1.3

 

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: CLP EU GHS (Regulation (EC) No 1272/2008
Conclusions:
Based on the results of a fully valid Local Lymph node assay (OECD 429, GLP), n-Butyl 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 n-Butyl methacrylate (99.88%) dissolved in acetone : olive oil (4 +1) 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).  n-Butyl methacrylate, three groups each of five female mice were treated daily with the test item at concentrations of 25, 50, and 100% (w/w) in acetone:olive oil (4+1) 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)) 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, all test item treated animals showed an erythema of the ear skin (Score 1). On days 4 and 5 the animals treated with 25 and 50% of n-Butyl methacrylate 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 2). Furthermore, on day 6 the animals treated with a test item concentration of 50 and 100% showed an ear skin erythema Score 1.

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 2.19, 3.28, and 5.41 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1). A clear dose response was observed. The EC3 value was calculated, to be 43.6% (w/v).

Therefore, n-Butyl 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:

Data availability

Several sensitization tests in Guinea pigs are available, including a modern guideline study according to the maximization protocol. Furthermore a new Local Lymph Node Assay (LLNA) in mice according to OECD 429 is available (MPA, 2013). In addition, human case studies have been published with patch test data in humans.

Animal experiments

The dermal sensitization study with n-Butyl methacrylate (99.88%) dissolved in acetone : olive oil (4 +1) 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).  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) 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)) 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.

In the course of the study no cases of mortality and no signs of systemic toxicity were observed.

On day 3, all test item treated animals showed an erythema of the ear skin (Score 1). On days 4 and 5 the animals treated with 25 and 50% of n-Butyl methacrylate 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 2). Furthermore, on day 6 the animals treated with a test item concentration of 50 and 100% showed an ear skin erythema Score 1.

In this fully valid study Stimulation Indices of 2.19, 3.28, and 5.41 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1). A clear dose response was observed. The EC3 value was calculated, to be 43.6% (w/v).

Therefore, n-Butyl methacrylate was found to be a skin sensitiser when tested in the Local Lymph Node Assay according to OECD TG 429 (MPA, 2013).

The delayed contact hypersensitivity of n-butyl methacrylate was evaluated in Guinea pigs according to OECD N°406 guideline (Magnusson and Kligman test) (CIT, 2008). The induction phase has been realized both by intradermal route on day 1 (5% in corn oil) and by cutaneous route on day 8 (50% (w/w) in ethanol/purified water (80/20, w/w)) in 1 group of guinea pigs (2 males and 3 females). The challenge phase was realized on day 22 by cutaneous application of n-butyl methacrylate (10% (w/w) in acetone ) on the right flank (vehicle on the left flank); the cutaneous reactions were scored 24 and 48 hours after the challenge phase. After the challenge application, no cutaneous reactions were noted on the left flank (application of the vehicle) of the animals. On the right flank (application of n-butyl methacrylate) of the animals, a discrete or moderate erythema was noted in 4/5 and 2/5 animals at the 24 and 48-hour readings, respectively. An oedema and dryness of the skin were also noted in 1/5 and 2/5 animals, respectively, at the 48-hour reading. In conclusion, n-butyl methacrylate induced delayed contact hypersensitivity in 4/5 (80%) guinea pigs.

In a study performed with 19 male guinea pigs, each animal received 100 mg of FCA in the four foot pads on day 0 (Chung and Giles, 1977). On days 0, 2 and 5 each animal received topical applications of 0.038 ml n-BMA in 95 % ethanol (volume applied: 0.2 ml). A first challenge was made on day 25 using a topical application of 2 or 5 % n-BMA in 95 % ethanol (volume applied: 0.05 ml). A second challenge was made on day 60 using either a topical challenge with 10 % n-BMA in olive oil or an intradermal challenge with either 0.01 ml or 0.1 ml of n-BMA in 0.1 ml saline. A third challenge was made on day 122 using either 0.4 or 5 % n-BMA in olive oil (volume applied: 0.05 ml). Macroscopic skin reactions (erythema and oedema) were evaluated and scored at 24, 48, 72, 96, 144 or 168 hours after each challenge. No reactions were seen after the first challenge which the authors attribute to the rapid evaporation of n-BMA from the skin surface. At the second challenge, 38 and 54 % of the animals challenged by intradermal injection with 0.01 and 0.1 ml n-BMA respectively gave positive reactions at 48 hours. Topical application at the second challenge resulted in positive reactions in 88 % of the animals. At the third challenge, 93 % of animals challenged with either 0.4 or 5 % n-BMA gave positive results at 72 hours after challenge. Nine male guinea pigs received 100 mg of FCA in the four foot pads on day 0. Each animal received also a single topical application of 0.0077 ml n-BMA in 0.2 ml of olive oil. On day 60 each received a topical application of 2 % or 5 % n-BMA in olive oil (volume applied 0.05 ml). On day 95 each received a topical application of 10 % n-BMA in olive oil. All animals gave positive responses at both application times.

A recently developed in vitro testing battery that addresses different stages of the sensitization process was employed on three of the members of the category – MMA, EMA and n-BMA. The battery addressed protein reactivity, activation of the Keap-1/Nrf2 signaling pathway and dendritic cell activation consisting out of the following assays: direct peptide reactivity assay (DPRA), LuSens, h-CLAT, and MUSST. The three esters were broadly positive in all three assays demonstrating the potential for contact allergy (Wiench et al., 2013)

Human data

Maibach et al., reported that in 542 dermatitis patients given covered patch tests with 1% n-BMA in petrolatum, one individual responded to n-BMA (Maibach et al. 1978). Six out of 243 contact dermatitis patients given 24-hr covered patch tests with n-BMA at a concentration of 2 % in petrolatum (Kanerva et al. 1997). Schnuch reported the prevalence of positive clinical challenge responses in dental clinicians that had been referred with dermatitis and suspected of having allergy to (meth)acrylates as 0.3% (1/347) for BMA (IVDK (Information Network of Dental Clinics) database; Schnuch, 1997). This author also reported the prevalence in a similar, pre-selected clinical cohort as 0.8% (9/1161) for MMA and 0.3% (2/625) for EMA (Schnuch, 1997). The prevalence of positive clinical challenge tests in patients referred with dermatitis with previous contact with (meth)acrylates was reported as 0.6% (2/331) for BMA (Tucker and Beck, 1999). n-BMA has frequently been included in the test substance lists of patients with known contact to (meth)acrylates, but response rates were generally lower than with MMA or EMA. It is not entirely clear, though, whether that is due to lower potency or lower exposure.

Summary

n-BMA is a skin sensitiser of weak potency. The methacrylate ester is a direct acting Michael acceptor electrophile, and on that basis is identified as potential contact allergen in relevant SAR models and the OECD (Q)SAR tool box (Bausch et al., 2011). nBMA is also positive in the in vitro DPRA and LuSens assays and in the MUSST (Kolle, S. 2013; Wiench et al., 2013).

Nevertheless, the primary acid metabolite, MAA, and the respective alcohol, are not skin sensitising in animal studies, so metabolism (hydrolysis) is a detoxification mechanism that occurs within the skin.

Migrated from Short description of key information:
n-Butyl methacrylate is a skin sensitizer.

Respiratory sensitisation

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

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

Justification for classification or non-classification

Based on the available data on n-butyl methacrylate, it is considered that the substance comprises the potential for skin sensitisation and has therefore to be classified with

R43/ skin sensitiser under 67/548/EEC

Cat. 1 skin sensitiser according to CLP (1272/2008/EC) and

Cat 1B, skin sensitiser according to UN GHS requirements

Based on the available data there is no evidence that n-BMA causes respiratory sensitisation. Therefore, a classification for respiratory sensitisation is considered as not justified.