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Description of key information

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
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:
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 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: 7-8 weeks (beginning acclimatisation)
- Weight at study initiation: mean weight: 18.6 ±0.9 g
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: At least 5 days. 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: 30-72 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artifical 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 %
Test concentrations (non-GLP) pre-tests: 12.5, 25, 50 and 100%
No. of animals per dose:
Main study: 4 females (nulliparous and non-pregnant)
Pre-tests (non-GLP): 2 females
Details on study design:
RANGE FINDING TESTS: (non-GLP)
- Compound solubility: in water: approx. 4000 mg/L at 25°C
- Irritation: no irritation effects were observed at test concentrations (12.5, 25, 50 and 100%) after single application.

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 different test item
concentrations of 25, 50 and 100% (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 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 81.0 µCi/ml ³HTdR (corresponds to
20.3 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 Na-thiopental (Trapanal, Altana,
78467 Konstanz, Germany).
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 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 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 (week day) from experimental start to necropsy.
Body weights: prior to the first application and prior to treatment with ³HTdR.
Clinical signs (local / systemic): once daily (week day). Especially the treatment sites were observed carefully.

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Results of the GLP Positive Control

Experiment performed in December 2006.
Positive control substance: alpha-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4+1)

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 1.91, 0.86, and 1.07 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 none of the tested concentrations induced an S.I. 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-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

128.20

---

---

---

---

---

BG II

88.65

---

---

---

---

---

CG1

3733.24

3624.8

8

453.1

 

25

2

7034.24

6925.8

8

865.7

1.91

50

3

3233.23

3124.8

8

390.6

0.86

100

4

3972.43

3864.0

8

483.0

1.07

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, 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 with3HTdR (g)

1

1

18.8

19.7

2

1

18.7

19.3

3

1

18.6

19.0

4

1

18.1

19.4

5

2

18.0

19.5

6

2

18.6

19.2

7

2

18.7

19.3

8

2

19.6

20.5

9

3

17.1

17.4

10

3

18.3

19.2

11

3

18.1

20.9

12

3

17.7

18.8

13

4

18.3

19.5

14

4

19.2

20.2

15

4

21.0

20.4

16

4

18.0

19.5

Mean

18.6

19.5

Standard Deviation

0.9

0.8

Results of the GLP Positive Control

Experiment performed in December 2006.

Positive control substance: alpha-Hexylcinnamaldehyde

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

197.50

---

---

---

---

---

BG II

22.03

---

---

---

---

---

CG1

4049.44

3939.7

8

492.5

 

5

2

8152.94

8043.2

8

1005.4

2.04

10

3

24974.40

24864.6

8

3108.1

6.31

25

4

49155.30

49045.5

6

6130.7

12.45

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)

2.04(a)

Group 3

10 (c)

6.31(d)

EC3 = (a-c) [(3-d)/(b-d)] + c = 6.1%(w/v)

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Ethyltriglycol methacrylate was not a skin sensitiser under the described conditions according to OECD 429 (Skin Sensitisation: Local Lymph Node Assay).
Executive summary:

In a dermal sensitization study with Ethyltriglycol methacrylate (92.6%, reactive ester content: 99.1%) dissolved in acetone : olive oil (4 +1) as a vehicle, 16 (4 per dose group) 7 -8 week old female CBA/CaOlaHsd mice were tested at concentrations of 25, 50, and 100 % (w/v) using the method of OECD 429 (Local Lmyphnode Assay). 

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

In the course of the study no cases of mortality were observed. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 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.

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 of ³HTdR 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 1.91, 0.86, and 1.07 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 this study, Ethyltriglycol methacrylate was therefore, found not to be 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Two relevant, reliable (Klimisch score 1; reliable without restrictions) studies are available for the sensitising potential of Ethyltriglycol methacrylate.

In this dermal sensitization study with Ethyltriglycol methacrylate dissolved in acetone: olive oil (4 +1) as a vehicle, 16 (4 per dose group) 7 -8 week old female CBA/CaOlaHsd mice were tested at concentrations of 25, 50, and 100 % (w/v) using the method of OECD 429 (Local Lmyphnode Assay). 

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

In the course of the study no cases of mortality were observed. No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. 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.

In this study Stimulation Indices of 1.91, 0.86, and 1.07 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 this study, Ethyltriglycol methacrylate was therefore, found not to be a dermal skin sensitizer under the described conditions.

Based on the results of this LLNA study (RCC Cytotest Cell Research GmbH, 2007) Ethyltriglycol methacrylate is not considered to be a skin sensitizier.

This result is supported by the results of the Guinea pig maximization test (Magnusson and Kligman method) according to OECD 406 (C.I.T. Centre International de Toxicologie, 1991) with Ethyltriglycol methacrylate where no skin sensitisation has been observed.



Migrated from Short description of key information:
Two relevant, reliable (Klimisch score = 1) and adequate studies are available. Based on the results of this LLNA study (RCC Cytotest Cell Research GmbH, 2007) and the supporting Guinea pig maximization test of Magnusson and Kligman according to OECD 406 (C.I.T. Centre International de Toxicologie, 1991) Ethyltriglycol methacrylate is not considered to be a skin sensitizier.

Justification for selection of skin sensitisation endpoint:
Guideline study OECD 429, GLP, negative
supporting study OECD 406, GLP, negative

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Justification for selection of respiratory sensitisation endpoint:
Inhalation is no relevant route of exposure.

Justification for classification or non-classification

Based on the results of the available LLNA study (Klimisch score: 1, RCC Cytotest Cell Research GmbH, 2007) and the supporting Guinea pig maximization test of Magnusson and Kligman according to OECD 406 (Klimisch score: 1, C.I.T. Centre International de Toxicologie, 1991) Ethyltriglycol methacrylate is not considered to be a skin sensitizier. Hence, Ethyltriglycol methacrylate is not classified as a skin sensitizier according to regulation (EC) 127/2008 and the former European directive on classification and labelling 67/548/EEC.