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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:
guideline study
Remarks:
GLP.
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)
Specific details on test material used for the study:
- Name of test material (as cited in study report): N-Dimethylaminopropyl methacrylamide
- Substance type: organic
- Physical state at room temperature: liquid
- Stability under test conditions: Stability in water: Several days at room temperature, refrigerated and in the freezer
- Storage condition of test material: At room temperature
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: 18.9 ±0.8 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: miscrible at 20°C
- Irritation: no irritation effects were observed at test concentrations (12.5, 25, 50 and 100%) after single application.
- 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). 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.

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). 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
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 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 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
Value:
25.18
Test group / Remarks:
25%
Parameter:
SI
Value:
29.79
Test group / Remarks:
50%
Parameter:
SI
Value:
43.38
Test group / Remarks:
100%
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, 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

128.20

---

---

---

---

---

BG II

88.65

---

---

---

---

---

CG1

3733.24

3624.8

8

453.1

 

25

2

91371.50

91263.1

8

11407.9

25.18

50

3

108086.00

107977.6

8

13497.2

29.79

100

4

157356.00

157247.6

8

19655.9

43.38

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 above 3.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

Until the second application of the test item no reddening of the ears was observed for any of the tested concentrations. After the third application of the test item slight reddening of the ears was observed at the lowest tested concentration (25%). The animals treated with 50 and 100% showed excessive local irritation of the ear skin after the third application.

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

20.2

19.8

6

2

18.5

19.3

7

2

17.7

19.5

8

2

18.3

19.6

9

3

18.6

19.4

10

3

20.0

20.7

11

3

19.1

19.4

12

3

19.6

19.0

13

4

18.2

18.3

14

4

20.6

20.0

15

4

19.3

19.9

16

4

18.5

19.8

Mean

18.9

19.5

Standard Deviation

0.8

0.5

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(b)

Group 3

10 (c)

6.31(d)

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

Interpretation of results:
sensitising
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The test item N-Dimethylaminopropyl methacrylamide was found to be a skin sensitiser under the described conditions.
Executive summary:

In a dermal sensitization study with N-Dimethylaminopropyl methacrylamide (99.22%) dissolved in acetone : olive oil (4 +1) as a vehicle, 16 (4per dode 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.

In the course of the study no cases of mortality were observed. Until the second application of the test item no erythema of the ears was observed for any of the tested concentrations. After the third application of the test item erythema of the ears occured in a concentration-dependent manner, slight at the lowest tested concentration (25%) and strong in the animals treated with 50 and 100%. Since the irritation effects did not occur at an earlier time point, it is likely to be caused by the sensitising effect.

In this study Stimulation Indices (S.I.) of 25.18, 29.79, and 43.38 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. Especially for the low dose (25%), where only a slight reddening was observed after the third application of the test item, it is unlikely that the high proliferative response of the lymph nodes was due to an irritating effect of the test item alone.

In this study, N-Dimethylaminopropyl methacrylamide is therefore, found to be a dermal skin sensitizer.

Based on the results of the available studies, N-[3 -(dimethylamino)propyl] methacrylamidehas to be classified as follows:

67/548/EC: Xi, R43, May cause sensitisation by skin contact. EG Regulation No. 1272/2008: Skin sensitisation Cat. 1, H317, may cause an allergic skin reaction.

 

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:
adverse effect observed (sensitising)
Additional information:

One relevant, reliable (Klimisch score 1; reliable without restrictions) study is available for the sensitising potential of

N-[3-Dimethylamino)propyl]methacrylamide.

In a dermal sensitization study with N-Dimethylaminopropyl methacrylamide (99.22%) 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 using the method of OECD 429 (Local Lymphnode 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. Until the second application of the test item no erythema of the ears was observed for any of the tested concentrations. After the third application of the test item erythema of the ears occured in a concentration-dependent manner, slight at the lowest tested concentration (25%) and strong in the animals treated with 50 and 100%. Since the irritation effects did not occur at an earlier time point, it is likely to be caused by the sensitising effect.

In this study Stimulation Indices (S.I.) of 25.18, 29.79, and 43.38 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. Especially for the low dose (25%), where only a slight reddening was observed after the third application of the test item, it is unlikely that the high proliferative response of the lymph nodes was due to an irritating effect of the test item alone.

In this study, N-Dimethylaminopropyl methacrylamide is therefore, found to be a dermal skin sensitizer.



Migrated from Short description of key information:
One relevant, reliable (Klimisch score = 1) and adequate study is available.
Date requirements for sensitising potential according Annex VII to X are fulfilled. Based on the results of this LLNA study (Evonik Industries AG,
2007) N-[3-Dimethylamino)propyl]methacrylamide was demonstrated to be a skin sensitiser in an OECD 429.

Justification for selection of skin sensitisation endpoint:
Guideline study OECD 429, GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no experimental or clinical evidence of respiratory sensitisation. In the absence of relevant findings N-[3-Dimethylamino)propyl]methacrylamide is not regarded as a respiratory sensitiser.


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 studies, N-[3 -(dimethylamino)propyl] methacrylamide has to be classified as follows:

67/548/EC: Xi, R43, May cause sensitisation by skin contact. EG Regulation No. 1272/2008: Skin sensitisation Cat. 1, H317, may cause an allergic skin reaction.