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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Nov 2006 - 06 Nov 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 429, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Remarks:
OECD Principles of GLP, as revised in 26 November 1997 [C(97)186/Final]
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Methacrylic acid ester 13.0
- Substance type: organic
- Physical state at room temperature: liquid
- Expiration date of the lot/batch: 10-Sep-2009
- Stability under test conditions:
- Storage condition of test material: At room temperature, light protected

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 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

Study design: in vivo (LLNA)

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.

Results and discussion

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)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.99
Test group / Remarks:
5 %
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:
SI
Value:
2.11
Test group / Remarks:
10 %
Remarks on result:
other:
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:
SI
Value:
2.66
Test group / Remarks:
25 %
Remarks on result:
other:
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)

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: Acetone:olive oil (4 +1)

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-

BGa)

number of lymph nodes

DPM per lymph nodeb)

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 with3HTdR (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-

BGa)

number of lymph nodes

DPM per lymph nodeb)

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.

Applicant's summary and conclusion

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.