2-Propenamide, N-(2,2-dimethoxyethyl)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-01 to 2010-01-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed study according to OECD Technical Guidelines.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

dated 24 April 2002

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificate attached to the report

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

N-(2,2-dimethoxyethyl)prop-2-enamide

EC Number:

610-472-5

Cas Number:

49707-23-5

Molecular formula:

C7 H13 N1 O3

IUPAC Name:

N-(2,2-dimethoxyethyl)prop-2-enamide

Details on test material:

- Sponsor's identification: NAAADA
- Container: plastic flask (n=1)
- Quantity: 51.80g (container and contents)
- Batch no: 700090909
- Production date: 09 September 2009
- CAS No of the main ingredients: 2226-96-2
- Date received: 16 September 2009
- Form: liquid
- Colour: yellow
- Storage: +4°C +/-4°C
- Expiry date: 09 January 2010
- Purity: >96%
- The item was used as supplied

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier (F-53941 Le Genest Saint Isle)
- Age at study initiation: 8 weeks
- Animals were nulliparous and non-pregnabt
- Weight at study initiation: 20.3 - 23.7g
- Housing: suspended solid-floor polypropylene cages furnished with soft woodflakes
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12hrs / 12hrs
- Animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

0%, 25%, 50%, 100%

No. of animals per dose:

4 females per dose

Details on study design:

PRELIMINARY STUDY
- Preliminary screening using one mouse
- The mouse was treated with 25µL of the undiluted test item
- Dorsal surface of each ear was treated on three consecutive days (d1, d2, d3)
- Daily observation of the treated animal on d1, d2, d3, d4, d5 and d6
- Any signs of toxicity or excessive local irriation noted during this period were recorded
- Body weight was recorded on day 1 prior to treatment and on day 6
- No systemic toxicity or excessive local reactions observed

MAIN STUDY
- Groups of four mice per each concentration
- Group 1: 4 females with vehicle only --> 0% of test item
- Group 2: 4 females with 25% test item
- Group 3: 4 females with 50% test item
- Group 4: 4 females with 100% test item
- An additional mouse was treated in each group in case of problems which may occur during the study, in particular during excision of lymph nodes
- Mice were treated by daily application of 25µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days
- Test item formulation was administered using an automatic micropipette and spread over the dorsal surface using the tip of the pipette

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Ear thickness and cell proliferation
- See specific details under: "Any other information on materials and methods incl. tables"

CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
- The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.4 compared to control values
- Any test item failing to produce a SI<1.4 will be classified as a "non sensitiser"
- % increase in ear thickness between day 1 and day 6

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

See attached tables (historical controls)

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

at 25 %

Value:

0.68

Variability:

pooled approach

Test group / Remarks:

4 animals

Results 2

Parameter:

SI

Remarks:

at 50 %

Value:

1.21

Variability:

pooled approach

Test group / Remarks:

4 animals

Results 3

Parameter:

SI

Remarks:

at 100 %

Value:

0.9

Variability:

pooled approach

Test group / Remarks:

4 animals

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Other method: cell counting and ear thickness

Any other information on results incl. tables

PRELIMINARY SCREENING TEST

Clinical observations, bodyweight and mortality data are given in Table 1 (attached).

No signs of systemic toxicity were noted.

Based on this information the dose levels selected for the main test were 25% (v/v), 50% (v/v) indimethylformamide and 100%.

CLINICAL OBSERVATIONS AND MORTALITY

Individual clinical observations and mortality data for test and control animals are given in Table 2 (attached).

No mortality and no signs of systemic toxicity were noted in the test and controls animals during thetest.

WEIGHT EVOLUTION

Individual bodyweights and bodyweight changes for test and control animals are given in Table 3 (attached).

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

ESTIMATION OF THE PROLIFERATIVE RESPONSE OF LYMPH NODE CELLS

The results of proliferation assay are given in Table 4 (given here and also attached).

Table 4: Cell count, Stimulation index and calculation of EC1.4

 

Groups	Test item	Cell count / groups (x10^6cells / mL)	Stimulation Index S.I.	Result	EC1.4value
1	DMF (Dimethylformamid)	23.80	n.a	n.a	n.a.
2	25%	16.28	0.68	Negative	n.a.
3	50%	28.83	1.21	Negative
4	100%	21.45	0.90	Negative

Stimulation Index = Cell count of treated group / Cell count of control group

EC1.4 value: Theorical concentration resulting in a SI value of 1.4

EC1.4=c + [(1.4 – d) / (b – d)] x (a – c)

Legend:                                                                                               

a = the lowest concentration giving stimulation index > 1.4

b = the actual stimulation index caused by a

c = the highest concentration failing to produce a stimulation index of 1.4

d = the actual stimulation index caused by c

No stimulation index of more than 1.4 was recorded for the three concentrations of the test item.

The Stimulation Index (SI) calculated by pooled approach was respectively 0.68, 1.21and 0.90 for thetreated group at 25%, 50% and 100%.

LOCAL IRRITATION

The results of ear thickness measurement and weight of skin biopsy are given in Tables 5 (attached) and 6 (attached).

No cutaneous reactions were observed at the concentrations of 25%, 50% and 100%.

No significant increase in ear thickness and in ear weight was recorded at the concentrations of 25%,50% and 100%. Therefore, the test item must be considered “non irritant” at the three concentrations.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Conclusions:

In view of these results, under these experimental conditions, the test item NAAADA is considered to be not a skin sensitiser in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the EEC Directives 67/548, 2001/59 and 99/45. No symbol and risk phrase are required. In accordance with the Globally Harmonized System (Regulation (EC) No. 1272/2008), the test item is not be classified. No signal word and hazard statement are required.

Executive summary:

The test was performed to assess the skin sensitisation potential of the test item NAAADA in the CBA/J strain mouse following topical applications to the dorsal surface of the ear.

Three groups of four animals, were treated for three consecutive days (D1, D2, D3) with 50 µL (25 µLper ear) of the test item as a solution in dimethylformamide (DMF) at concentrations of 25%, 50%(v/v) and 100%. A further group of four animals was treated with DMF.

On D6, the proliferation of lymphocytes in the draining auricular lymph nodes was determined by cellcounting. The experimental protocol was established accordingthe OECD Guideline n°429 datedApril 24^th, 2002 and the test method B. 42 of the council regulation No.440/2008.

No mortality and no signs of systemic toxicity were noted in the test and control animals during thetest.

No cutaneous reaction was observed.

No significant increase in ear thickness and in ear weight was recorded at the concentrations of 25%, 50% and 100%. Therefore, the test item must be considered “non irritant” at the three concentrations.

The Stimulation Index (SI) calculated by pooled approach was 0.68, 1.21 and 0.90 for the treatedgroups at 25%, 50%, and 100%, respectively.

In view of these results, under these experimental conditions, the test item NAAADA is considered to be not a skin sensitiser in accordance with the criteria for classification, packaging and labelling ofdangerous substances and preparations of theEEC Directives 67/548, 2001/59 and 99/45. Nosymbol and risk phrase are required.

In accordance with the Globally Harmonized System (Regulation (EC) No 1272/2008), the test item is not to be classified in category 1. No signal word and hazard statement are required.