Butyl methacrylate

Skin sensitisation

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

individual approach (adopted 22 July 2010)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(dated May 30, 2008)

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD Principles of GLP, as revised in 1997, ENV/MC/CHEM(98)17)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl methacrylate

EC Number:

202-615-1

EC Name:

Butyl methacrylate

Cas Number:

97-88-1

Molecular formula:

C8H14O2

IUPAC Name:

butyl methacrylate

Details on test material:

- Name of test material (as cited in study report): n-Butyl methacrylate (CAS: 97-88-1)
- Substance type: organic
- Physical state at room temperature: liquid
- Storage condition of test material: At room temperature

In vivo test system

Test animals

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.

Study design: in vivo (LLNA)

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.

Results and discussion

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.

In vivo (LLNA)

Results

Results 1

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.

Results 2

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

Any other information on results incl. tables

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 with^3HTdR (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

 

Applicant's summary and conclusion

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%)

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