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Skin sensitisation

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
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
Type:
Constituent

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: 7 weeks (beginning of acclimatisation)
- Weight at study initiation: mean weight: 17.3 - 21.3 g
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Batch no. 48/06 Provimi Kliba AG, CH-4303 Kaiseraugst)
- Water (e.g. ad libitum): community tap water from Itingen, ad libitum
- Acclimation period: 7 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): 22 ±3 °C
- Humidity (%): relative humidity: 30-70 %
- Air changes (per hr): 10 - 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour dark / artifical light: 12 hour with 8 hours music during the light period

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Test concentrations (main study): 0 (vehicle group), 2.5, 5.0 10 %
Test concentrations (non-GLP) pre-tests: 25, 50 and 100% (undiluted)
No. of animals per dose:
Main study: 4 females (nulliparous and non-pregnant)
Pre-tests (non-GLP): 1 female
Details on study design:
RANGE FINDING TESTS: (non-GLP)
- Compound solubility: Acetone/olive oil (4+1, v/v) was selected as the suitable vehicle and used to the main test.
- Irritation: In a non-GLP local toxicity pre-test, concentrations used in the main study were determined following treatment of three single animals
with one of three different concentrations: 25 %, 50 % and 100 % (undiluted), on both ears on three consecutive days. One day after each topical
application, slight to severe ear erythema and/or moderate ear swelling were observed at the dosing sites of 100 % (undiluted). One day after the
second and the third topical application, slight or moderate ear erythema andlor slight to moderate ear swelling were observed at the dosing sites of 25 % and 50 %.
The pre-test results determined that 10 % (w/v) was the highest dosing concentration to be used to the main tests for avoiding severe local irritation and/or possible systemic toxicity.
- Lymph node proliferation response:

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 2.5, 5.0 and 10% (w/v) in acetone : olive oil (4 +1, v/v). 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 Amersham Biosciences UK Limited, Buckinghamshire England (Amersham product code no.
TRA 310, aqueous solution, sterilized 74 GBq/mmol 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 phosphate-buffered saline (PBS) containing 78.84 µCi/ml ³HTdR
(corresponds to 19.7 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 inhalation of CO2 (dry ice).
The draining lymph nodes were rapidly excised and pooled in 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 glass scintillation vials with 10 ml of ‘Irga-Safe Plus’ 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).

Observations
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Once daily from acclimatization start to the termination of in-life phase.
Body weights: On the test day 1 (prior to the first application) and on the test day 6.
Clinical signs (local / systemic): Once daily on the day of animal delivery and from test day 1 (after the first application) to the termination of
in-life phase.
Size of the draining lymph nodes: Immediately after the excision, the size of nodes from test item groups was compared with the size of nodes
from control group.

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 August 2006.
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
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices (S.I.) of 0.9, 0.9 and 1.9 were determined with the test item at concentrations of 2.5, 5.0, and 10% in acetone: olive oil (4+1, v/v), 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, 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

9

---

---

---

---

---

BG II

6

---

---

---

---

---

CG1

5298

5290

8

661

 ---

2.5

2

4684

4676

8

585

0.9

5

3

4533

4525

8

566

0.9

10

4

10061

10053

8

1257

1.9

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 not above 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

Neither clinical / local signs nor other findings were observed in any animals of the control group, Group 2 (2.5 %) or Group 3

(5 %). One day after the first topical application, slight ear erythema was observed at both dosing sites in all mice of Group 4

(10 %), persisting for a total of two days.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with3HTdR and prior to sacrifice, 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

19.8

20.5

2

1

17.3

18.3

3

1

17.7

18.9

4

1

18.6

19.3

5

2

20.5

23.2

6

2

20.3

22.2

7

2

20.0

20.8

8

2

19.9

21.1

9

3

19.6

20.6

10

3

18.5

19.6

11

3

20.2

21.5

12

3

18.9

21.4

13

4

16.8

18.8

14

4

18.8

20.7

15

4

18.4

19.2

16

4

19.3

20.4

SIZE OF THE DRAINING LYMPH NODES

No abnormal findings were observed in the size or other physical features of the draining lymph nodes.

Results of the GLP Positive Control

Experiment performed in August 2006.

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

25

---

---

---

---

---

BG II

28

---

---

---

---

---

CG1

4159

4132

8

517

 ---

5

2

10051

10024

8

1253

2.4

10

3

10532

10505

8

1313

2.5

25

4

37334

37307

8

46663

9.0

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 3

10 (a)

2.5(b)

Group 4

25 (c)

9.0(d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 11.2%(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 lying immediately below and above 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 0.9, 0.9 and 1.9 were determined with the test item at concentrations of 2.5 %, 5 % and 10 % (w/v), respectively, in acetone/olive oil
(4/1, v/v).
Based on the test results, the test item Methacrylic acid ester 17.4 does not show an allergenic potential when tested up to the concentration of
10 % (wlv) in acetone/olive oil (4/1 , v/v).
Executive summary:

In a dermal sensitization study with Methacrylic acid ester 17.4 (96.84%) dissolved in acetone : olive oil (4 +1, v/v) as a vehicle, 16 (4 per dose group) 7 week 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 2.5 %, 5 % and 10 % (w/v) in acetone/olive oil (4/1, v/v) 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, v/v) only.

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

In the course of the study no cases of mortality were observed.

Neither clinical / local signs nor other findings were observed in any animals of the control group, Group 2 (2.5 %) or Group 3 (5 %). One day after the first topical application, slight ear erythema was observed at both dosing sites in all mice of Group 4 (10 %), persisting for a total of two days.

In this study Stimulation Indices (S.I.) of 0.9, 0.9, and 1.9 were determined with the test item at concentrations of 2.5, 5.0, and 10% in acetone : olive oil (4 +1, v/v), respectively. No dose-response relationship was observed. The EC3 value could not be determined because this calculation requires a S.I. value of greater than 3.

In this study, Methacrylic acid ester 17.4 is therefore, not a dermal skin sensitizer.

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