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Description of key information

Methacrylic anhydride was sensitising to skin in an OECD 429 Local Lymphnode assay.

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:
26.01.2007 - 16.08.2007
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 7-8 weeks
- Housing: single
- Diet (e.g. ad libitum): pelleted standaard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen
- Water (e.g. ad libitum): tap water ad libitum
- Initial Weight: mean initial weight 18.5 g
- Acclimation period: Under the test conditions after health xamination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 30-72 %
- Photoperiod (hrs dark / hrs 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:
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 j.J1, was spread over the entire dorsal surface (0 - 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 IJI of 80.5 j.JCi/ml 3HTdR (corresponds to 20.13 j.JCi 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 Na-thiopental (Trapanal, Altana, D-78467 Konstanz). The draining Iymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled Iymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffe red saline (approx. 10 ml) the Iymph 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 and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a ß-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The ß-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Interpretation of Raw Data
The proliferative response of Iymph node cells is expressed as the number of radioactive disintegrations per minute per Iymph node (DPM/node) and as the ratio of 3HTdR incorporated into Iymph node cells of test Iymph nodes relative to that recorded for control Iymph nodes (stimulation index). Before DPM/node values were deter.mined, 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.
The decision to select a STIMULATION INDEX (S.I.) of 3 as an arbitrary indication of sensitizing activity was made on the basis of investigations performed with a wide range of chemicals.

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)n from experimental start to necropsy.
Body weights: Prior to the first application and prior to treatment with ³HTdR.
Clinical signs (Iocal systemic): Once daily (week day) during the application of the test item. 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
Study performed in December 2006.
Positive control substance: a-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1)


Test itern
Concentration
% (w/v)
Group
Measurement
DPM Calculation Result
DPM-BG a) Number of lymph nodes DPM per lymph node S.I.
- BG1 197.50 - - - -
- BG2 22.03 - - - -
- CG1 4049.44 3939.7 8 492.5
5 TG1 8152.94 8043.2 8 1005.4 2.04
10 TG2 24974.40 24864.6 8 3108.1 6.31
25 TG3 49155.30 49045.5 8 6130.7 12.45


BG = Background (1 ml 5% trichloroacetic acid) in duplicate
CG = Control Group
TG = Test Group
S.I. = Stimulation Index
a) = The mean value was taken from the figures BG land BG 11
b) = Since the Iymph nodes of the an im als of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of Iymph nodes

Test item concentration %
(w/v) S.I.
Group 1 5 (a) 2.04 (b)
Group 2 10 ‘(c) 6.31 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 6.1% (w/v)

Parameter:
SI
Value:
12.6
Test group / Remarks:
25%
Parameter:
SI
Value:
13.69
Test group / Remarks:
50%
Parameter:
SI
Value:
14.57
Test group / Remarks:
100%
Parameter:
EC3
Remarks on result:
not determinable

Calculation and Results of Individual Data

Test itern

Concentration

% (w/v)

 

Group

 

Measurement

DPM

Calculation

Result

DPM-BG a)

Number of lymph nodes

DPM per lymph node

S.I.

-

BG1

21.22

-

-

-

-

-

BG2

15.74

-

-

-

-

-

CG1

6372.35

6353.9

8

794.2

 

5

TG1

76632.90

76614.4

8

9576.8

12.06

10

TG2

87015.30

86996.8

8

10874.6

13.69

25

TG3

92598.50

92580.0

8

11572.5

14.57

 

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

CG=Control Group

TG=Test Group

S.I.=Stimulation Index

a)=The mean value was taken from the figures BG land BG11

b)=Since the Iymph nodes of the an im als of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of Iymph nodes

ViabilityIMortality

No deaths occurred during the study period.

 

Clinical Signs

The animals did not show any clinical signs of toxicity after the first and second application. After the third application the mid (50 %) and the high dose (100 %) induced redness of the ear skin of all four animals of the group.

 

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

 

Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The test item Methacrylic anhydride was found to be a skin sensitiser. The EC3 value could not be calculated, since all S.I.'s were above 3. CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1A
Executive summary:

In a dermal sensitization study with Methacrylic anhydride (95.12%) dissolved in acetone : olive oil (4 +1) as a vehicle, 16 (4per dose group) 7 -8 week old female CBA/CaOlaHsd mice were tested using the method of OECD 429 (Local Lymphnode Assay). The validation-/positive control experiment was performed with alpha-Hexyl cinnamic aldehyde.

The animals did not show any clinical signs of taxi city after the first and second application. After the third application the mid (50 %) and the high dose (100 %) induced redness of the of the ear skin of all four animals of the group. No cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 12.06, 13.69 and 14.57 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 SI´s were above 3. The effect was due to the sensitising potential of the test item even though reddening of the ears occurred at the mid and high dose. Even at the lowest tested concentration where no redness of the ears was observed, the S.I. was well above the threshold for sensitisers.

For the evaluation of the effect strength however, also the acidic properties of MAA as hydrolysis product and primary metabolite of MAAH have to considered.

CLP EU GHS (Regulation (EC) No 1272/2008) classification: sensitizing category 1A

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

In a mouse local lymphnode assay acc. OECD 429 methacrylic anhydride was found to be sensitising to skin Stimulation Indices (S.I.) of 12.06, 13.69, and 14.57 were determined with the test item at concentrations of 25, 50, and 100 % in acetone: olive oil (4 + 1), respectively). For the evaluation of the effect strength, the acidic properties of MAA as hydrolysis product and primary metabolite of MAAH have to considered.

Justification for selection of skin sensitisation endpoint:
One GLP Guideline study available with Klimisch score 1.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Methacrylic anhydride fulfils the criteria for skin sensitising category 1 according to CLP (1272/2008/EC) and UN GHS.