2-Propenoic acid, 2-methyl-, C6-12-alkyl esters

Endpoint summary

Description of key information

The conclusion of Kimber and Pemberton that MMA and the lower methacrylate esters showing a weak skin sensitizing potency can be directly adopted to the methacrylic esters having slightly longer alcohol chains. The expected trend of decreasing potency with increasing ester size was obviously superimposed by other mechanism, therefore this assumption of a weak sensitizing potency has also to be stated for the closely related NUMA constituents ranging from C9-C14 methacrylatesleading to a classification with skin sens. 1B..

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Method in accordance with OECD Guideline, GLP.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Principles of method if other than guideline:

other: modified Buehler-test (1965)

GLP compliance:

yes

Type of study:

Buehler test

Justification for non-LLNA method:

The study was conducted before the LLNA was implemented in the OECD guideline.

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

Body weight 336-428 grams obtained from Davidson Mill Farm, Jamesburg, NJ. Aniamls individually identified by color coding.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.4 ml; the first induction application was a 20% dilution in water ; the second and third induction applications was 15% in water. The challenge
application was 10% in water.

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

0.4 ml; the first induction application was a 20% dilution in water ; the second and third induction applications was 15% in water. The challenge
application was 10% in water.

No. of animals per dose:

20: test substance
10: vehicle control
5: positive control
5: naive positive control

Details on study design:

A group of twenty animals was closely clipped over the induction site on their left flank one day prior to intiation and repeated as necessary.
There were three induction applications

Positive control substance(s):

yes

Remarks:

99+% DNCB

Positive control results:

The positive control (DCNB) induced and challenged as an 0.1% w/v concentration in a 50% ethanol: 0.9% saline solution is a skin sensitizer in albino
guinea pigs. After challenge application of the positive control article, no dermal irritation was observed for the naive postive control animals.

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% in deionized water challenge concentration

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10% in deionized water challenge concentration. No with. + reactions: 0.0. Total no. in groups: 20.0.

Test article group: no dermal irritation to eschar was observed during the induction phase of the study. After the challenge application no redness was observed other than slight patchy redness exhibited at 48 hrs by 2/20 animals. The incidence of sensitization was 0/20 and the severity was 0.0, 0.5 and 0.0 for 24, 48 and 72 hours, respectively.

Vehicle control group:

No dermal irritation to eschar was observed during the induction phase of the study. After the challenge application no redness was observed other than slight patchy redness exhibited at 48 hrs by 2/10 animals, and 3/10 animals at 72 hours. The vehicle site did not exhibit any redness.

Positive control group: see above

Naive positive control group: see above

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In a valid guideline study, MAA was not a dermal sensitizer in albino guinea pigs. Methacrylic Acid did appear to be a skin irritant at
concentrations of 15% and above.
It was also concluded that a 0.1% w/v concentration of DNCB in a 50% ethanol: 0.9% saline solution produced delayed contact hypersensitivity in
albino guinea pigs.

Executive summary:

In a valid guideline study, MAA was not a dermal sensitizer in albino guinea pigs. Methacrylic Acid did appear to be a skin irritant at concentrations of 15% and above.

It was also concluded that a 0.1% w/v concentration of DNCB in a 50% ethanol: 0.9% saline solution produced delayed contact hypersensitivity in albino guinea pigs.

Reference 2

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

Guideline:

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

Version / remarks:

individual approach (adopted 22 July 2010)

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)

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): 19.8 - 25.0 g (mean)
- Housing: group, Makrolon Type II (pre-test) / III (main sudy), with wire mesh top
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water (e.g. ad libitum): 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/w)
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:

In order to study a possible allergenic potential of 2-Ethylhexyl methacrylate, 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) 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 different 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 78.3 µCi/ml 3HTdR (corresponds to 19.6 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 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)
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.

Parameter:

SI

Remarks on result:

other: Test substance: 25 % S.I.=1.53 50 % S.I.=2.66 100 % S.I.=2.85 EC3 value: not determined A clear dose response was observed.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control group (negative control with vehicle, only): 128.9 DPM per lymph node (2 lymph nodes) Test substance: 25% DPM/lymph node: 196.9 50% DPM/lymph node: 342.3 100% DPM/lymph node: 367.1

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	22	---	---
---	---	BG II	25	---	---
0	1	1	108	84.5	---
0	1	2	176	152.5	---
0	1	3	99	75.5	---
0	1	4	129	105.5	---
0	1	5	250	226.5	---
25	2	6	239	215.5	1.7
25	2	7	208	184.5	1.4
25	2	8	282	258.5	2.0
25	2	9	240	216.5	1.7
25	2	10	133	109.5	0.8
50	3	11	299	275.5	2.1
50	3	12	295	271.5	2.1
50	3	13	454	430.5	3.3
50	3	14	347	323.5	2.5
50	3	15	434	410.5	3.2
100	4	16	333	309.5	2.4
100	4	17	283	259.5	2.0
100	4	18	373	349.5	2.7
100	4	19	442	418.5	3.2
100	4	20	522	498.5	3.9

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	22	---	---		---
---	BG II	25	---	---		---
---	CG1	---	128.9	62.2		1.0
25	2	---	196.9	55.5		1.53
50	3	---	342.3	74.6		2.66
100	4	---	367.1	93.7		2.85

 

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 could not be calculated, since all SI´s are below 3.

 

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No systemic findings were observed during the study period. On day 4 the animals treated with a concentration of 50% showed an erythema of the ear skin (Score 1) and animals treated with 100% of Ethylhexyl methacrylate showed an erythema of the ear skin (Score 2). On days 5 and 6 the ear skin erythema of the 100% dose group decreased to 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	24.2	22.4
2	1	23.4	24.2
3	1	23.0	23.0
4	1	19.8	20.2
5	1	20.7	19.7
6	2	22.8	22.8
7	2	20.7	22.0
8	2	21.3	22.2
9	2	20.6	21.7
10	2	21.3	21.1
11	3	23.8	23.4
12	3	22.0	21.1
13	3	24.8	24.0
14	3	20.8	22.4
15	3	21.1	21.8
16	4	21.2	22.7
17	4	21.3	21.9
18	4	20.0	21.7
19	4	25.0	23.3
20	4	23.6	24.6
	Mean	22.1	22.3
	Standard Deviation	1.6	1.3

 

Interpretation of results:

not 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), 2-Ethylhexyl methacrylate is not considered to be a skin sensitizer.
CLP EU GHS (Regulation (EC) No 1272/2008) classification: not sensitizing

Executive summary:

In a dermal sensitization study with 2 -Ethylhexyl methacrylate (99.23%) 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).  2-Ethylhexyl 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 4 the animals treated with a concentration of 50% showed an erythema of the ear skin (Score 1) and animals treated with 100% of Ethylhexyl methacrylate showed an erythema of the ear skin (Score 2). On days 5 and 6 the ear skin erythema of the 100% dose group decreased to 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 1.53, 2.66, and 2.85 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.

Therefore, 2-Ethylhexyl methacrylate was not 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: not sensitizing

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 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Netherlands
B.V. Postbus 6174
5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: 19.3 - 21.9 g (mean)
- Housing: single
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
(Harlan Laboratories B.V., 5960 AD Horst / Netherlands)
- Water (e.g. ad libitum): 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 - 65%
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:

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

Concentration:

25 %, 50 % and 100 %

No. of animals per dose:

4

Details on study design:

In order to study a possible allergenic potential of Isodecyl methacrylate, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four 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 group. 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 -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 different test item concentrations of 25, 50, and 100% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (  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
3H-methyl thymidine (3HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310; specific activity, 2 Ci/mmol; concentration, 1 mCi/ml).
Five days after the first topical application, all mice were administered with 250 µl of 78.3 µCi/ml 3HTdR (corresponds to 19.6 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 intraperitoneal injection of Pentobarbital-Natrium (Release, WDT, D-30827 Garbsen).
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH, D-63110 Rodgau) and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a -scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau). Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The -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 node (DPM/node) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation index). Before DPM/node 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.
Clinical signs (local / systemic) In the pre-test clinical signs were recorded within 1 hour and 24 ± 4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ± 4 hours after the first and second application as well as on the day of preparation. 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.

Positive control results:

Results of the GLP Positive Control

Experiment performed in June 2009.
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 23 --- --- --- ---
--- BG II 19 --- --- --- ---
0 1 5842 5821 8 727.6
5 2 10450 10459 8 1303.6 1.79
10 3 12168 12147 8 1518.4 2.09
25 4 39834 39813 8 4976.6 6.84

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

Test item concentration % S.I.
Test Group 3 10 (a) 2.09 (b)
Test Group 4 25 (c) 6.84 (d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 12.9 % (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:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 1.43, 1.22 and 0.97 were determined with the test item at concentrations of 25, 50, and 100% in acetone : olive oil (4+1), respectively. The EC3 value could not be calculated, since all induced S.I.'s are below 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see below

Calculation and Results of Individual Data

 

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	21	---	---	---	---
---	BG II	31	---	---	---	---
---	CG1	4282	4256	8	523.0
25	2	6106	6080	8	760.0	1.43
50	3	5235	5209	8	651.1	1.22
100	4	4162	4136	8	517.0	0.97

 

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

CG1=   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

The EC3 value could not be calculated, since all SI´s are below 3.

 

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period

Body Weights

The body weight of the animals, recordedprior 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	21.1	21.5
2	1	20.0	20.9
3	1	19.7	20.1
4	1	21.9	22.6
5	2	21.5	23.2
6	2	20.8	22.3
7	2	21.1	21.4
8	2	21.6	23.5
9	3	19.7	20.6
10	3	19.7	21.1
11	3	21.0	21.4
12	3	20.2	20.0
13	4	19.8	21.3
14	4	20.7	21.4
15	4	19.3	20.0
16	4	20.8	20.2
	Mean	20.6	21.3
	Standard Deviation	0.8	1.1

 

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Isodecyl methacrylate was found to be not a skin sensitiser under the described conditions.

Executive summary:

In order to study a possible contact allergenic potential of Isodecyl methacrylate, three groups each of four 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 four 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 group. 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 ab-scintillation counter. All treated animals survived the scheduled study period and no signs of toxicity were observed. 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 1.43, 1.22, and 0.97 were determined with the test item at concentrations of 25, 50, and 100% in acetone:olive oil (4+1). The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

In conclusion, Isodecyl methacrylate was not a skin sensitiser when tested in the Local Lymph Node Assay according to OECD TG 429.

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 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

01 Nov 2006 - 06 Nov 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study OECD 429, GLP. Study according to relevant guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 24 April 2002)

Deviations:

no

Principles of method if other than guideline:

Three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application,
the mice were intravenously injected into a tail vein with radiolabelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous
injection, the mice were sacrificed and the draining auricular lymph nodes excised and pooled per group. 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.

GLP compliance:

yes (incl. QA statement)

Remarks:

OECD Principles of GLP, as revised in 26 November 1997 [C(97)186/Final]

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 Netherlands, B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of treatment)
- Weight at study initiation: mean weight: 20.0 ±0.9 g
- Housing: group (where relevant)
- Diet: ad libitum, pelleted standard diet (Harlan Laboratories GmbH, D-33178 Borchen)
- Water: ad libitum, tap water from Gemeindewerke D-64380 Rossdorf, Germany
- Acclimation period: 5 days prior to the first topical application under test conditions after health examination. Only animals without any visible
signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24 °C
- Humidity (%): relative humidity: 35-65 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour dark / artifical light: 12 hour

Vehicle:

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

Concentration:

Test concentrations (main study): 0 (vehicle group), 5, 10 and 25 %
Test concentrations ( pre-tests): 25, 50 and 100% (undiluted)

No. of animals per dose:

Main study: 4 females (nulliparous and non-pregnant)
Pre-tests: 2 females

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Acetone:olive oil (4+1) was selected as the suitable vehicle and used to the main test.
- Irritation: Two mice were treated with concentrations of 10 and 25% each on three consecutive days. In the pre-test clinical signs were recorded
within 1 hour and 24 ± 4 hours after each application as well as on day 7. At the tested concentrations the animals did not show any signs of irritation or systemic toxicity.
The test item in the main study was assayed at 5, 10 and 25 %. No severe irritant effects were tolerated choosing the test concentrations.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node assay
- Criteria used to consider a positive response:
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/node)
and as the ratio of ³HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (stimulation
index S.I.). Before DPM/node 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 S.I..
- 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.

The decision to select a Stimulation index (S.I.) of 3 as an arbitrary indication of sensitising activity was made on the basis of investigations performed with a wide range of chemicals.

TREATMENT PREPARATION AND ADMINISTRATION:

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 the test item
concentrations of 5, 10 or 25% (w/v) in acetone:olive oil (4+1). The application volume, 25 µl, was spread over the entire dorsal surface (diameter ca. 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 (³HTdR)
³H-methyl thymidine (³HTdR) was purchased from GE Healthcare (GE Healthcare product code no. TRA 310, specific activity, 2 Ci/mmol;
concentration 37 MBq/ml (1 mCi/ml) quatities: 9.25 MBq (250 µCi)
Five days after the first topical application, all mice were administered with 250 µl of 79.1 µCi/ml ³HTdR (corresponds to 19.8 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 intraperitoneal injection of Pentobarbital-Natrium (Release, WDT,
D-30827 Garbsen, Germany).
The draining lymph nodes were rapidly excised and pooled in phosphate buffered saline (PBS) for each experimental group (8 nodes per group).
Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh
size). After washing twice 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 plastic scintillation vials with 10 ml of ‘Ultima Gold’ scintillation liquid (Perkin Elmer (LAS) GmbH,
D-63110 Rodgau, Germany) and thoroughly mixed.
The level of ³HTdR incorporation was then measured on a beta-scintillation counter (Tricarb 2900 TR, Perkin Elmer (LAS) GmbH, D-63110 Rodgau,
Germany). 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).

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.
Clinical signs (local/systemic): In the pre-test clinical signs were recorded within 1 hour and 24 ±4 hours after each application as well as on day 7. In the main experiment clinical signs were recorded within 1 hour each application, and 24 ±4 hours after the first and
second application as well as on the day of preparation. 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.

Positive control results:

Results of the GLP Positive Control

Experiment performed in January 2009.
Positive control substance: alpha-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1, v/v)

table see: Remarks on results including tables and figures (results of the GLP positive control)

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

In this study Stimulation Indices (S.I.) of 0.99, 2.11 and 2.66 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4+1), respectively. No dose-response relationship was observed. The EC3 value could not be determined, since this calculation requires a S.I. value greater than 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see: Remarks on results including tables and figures (results of Individual data)

Calculation and Results of Individual Data

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	15	---	---	---	---
---	BG II	17	---	---	---	---
---	CG1	4535	4519	8	564.9	1.00
5	2	4496	4480	8	560.0	0.99
10	3	9560	9544	8	1193.0	2.11
25	4	12021	12005	8	1500.6	2.66

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

CG1=   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

The EC3 value could not be calculated, since all SI´s are below 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

Systemic signs of toxicity were not observed during the experiment. Local effects (ear reddening) were only observed with the highest treatment dose (25%) 24 hours after the second and 1 hour after the third application but not on day 6.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with³HTdR, were 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	19.2	20.6
2	1	20.1	20.7
3	1	20.9	21.5
4	1	20.3	21.9
5	2	20.9	23.0
6	2	19.6	20.4
7	2	19.6	20.5
8	2	18.9	19.5
9	3	18.1	20.4
10	3	19.2	20.6
11	3	21.1	22.5
12	3	21.3	23.0
13	4	20.7	21.9
14	4	19.9	20.9
15	4	20.8	21.4
16	4	19.5	21.5

Results of the GLP Positive Control

Experiment performed in January 2009.

Positive control substance: alpha-Hexylcinnamaldehyde

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

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	33	---	---	---	---
---	BG II	37	---	---	---	---
---	CG1	5392	5357	8	669.6	---
5	2	8026	7991	8	998.9	1.49
10	3	22385	22350	8	2793.8	4.17
25	4	26285	26250	8	3281.3	4.90

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

CG1=   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

	Test item concentration % (w/v)	S.I.
Group 2	5 (a)	1.49(b)
Group 3	10 (c)	4.17(d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 7.8%(w/v)

EC3 = Estimated concentration for a STIMULATION INDEX (S.I.) of 3.

a,b,c,d = Co-ordinates of the two pair of data immediatelylying above and below the S.I. value of 3 on the LLNA dose response

plot.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in
incorporation of 3HTdR compared with concurrent controls, as indicated by the S.I.
In this study S.I. of 0.99, 2.11 and 2.66 were determined with the test item at concentrations of 5%, 10%, and 25% (w/v) in acetone:olive oil (4+1),
respectively.
Based on the test results, the test item Methacrylic acid ester 13.0 does not show an allergenic potential when tested up to the concentration of
25 % (w/v) in acetone:olive oil (4+1).

Executive summary:

In a dermal sensitization study with Methacrylic acid ester 13.0 dissolved in acetone:olive oil (4 +1) as a vehicle, 16 (4 per dose group) 8 -12 weeks old female CBA/CaOlaHsd mice were tested using the method of OECD 429 (Local Lmyphnode Assay). 

Three groups each of four female mice were treated daily with the test item at concentrations of 5%, 10 %, and 25 % (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 four female mice was treated with the vehicle (acetone:olive oil (4 +1)) only.

The validation-/positive control experiment was performed with alpha-Hexyl cinnamic aldehyde in January 2009.

In the course of the study no cases of mortality were observed. Systemic signs of toxicity were not observed during the experiment. Local effects (ear reddening) were only observed with the highest treatment dose (25%) 24 hours after the second and 1 hour after the third application but not on day 6.

In this study Stimulation Indices (S.I.) of 0.99, 2.11 and 2.66 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4 +1), respectively. No dose-response relationship was observed. The EC3 value could not be determined since none of the tested concentrations induced an S.I. value greater than 3.

In this study, Methacrylic acid ester 13.0 is therefore, not a dermal skin sensitizer under the described conditions.

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 5

Endpoint:

skin sensitisation: in vitro

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included as attachment. Please also see attached justification.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The constituents of the UVCB substance NUMA (Nonyl-Undecyl methacrylate) are structurally related mono alkyl methacrylate esters differing only in the respective alcoholic moieties. The main proportion of these esters are of the n-type the minor proportion of the iso type. Considering the small amount of iso-types and the negligible differences in (eco-) toxicological properties between the n- and iso types, in this assessment both types of one ester with one specific chain length are assessed together as a whole.
Further information is included as attachment. Please also see attached justification Chapter 1

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Further information is included as attachment. Please also see attached justification Chapter 1.

3. ANALOGUE APPROACH JUSTIFICATION
Please see attached justification Chapter 1 and 3.

4. DATA MATRIX
Please see attached justification Chapter 1.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Parameter:

other: read across

Remarks:

Based on read across to analogues substances and/or constituents of the target UVCB substance it can be assumed that all constituents ranging from C9-C14 methacrylates and the UVCB NUMA, have weak sensitizing potency.

Remarks on result:

other: Based on read across to analogues substances and/or constituents of the target UVCB substance it can be assumed that all constituents ranging from C9-C14 methacrylates and the UVCB NUMA, have weak sensitizing potency.

Interpretation of results:

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

Conclusions:

Based on read across to analogues substances and/or constituents of the target UVCB substance it can be assumed that all constituents ranging from C9-C14 methacrylates and the UVCB NUMA, have weak sensitizing potency.

Executive summary:

The conclusion of Kimber and Pemberton that MMA and the lower methacrylate esters showing a weak sensitizing potency can be directly adopted to the methacrylic esters having slightly longer alcohol chains. The expected trend of decreasing potency with increasing ester size was obviously superimposed by other mechanism, therefore this assumption of a weak sensitizing potency has also to be stated for the closely related NUMA constituents ranging from C9-C14 methacrylates leading to a classification with skin sens. 1B..

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)