Registration Dossier

Administrative data

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: Data obtained from a guideline study according to OECD Guideline 429 and therefore considered reliable without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Acetamide
EC Number:
200-473-5
EC Name:
Acetamide
Cas Number:
60-35-5
Molecular formula:
C2H5NO
IUPAC Name:
acetamide
Test material form:
solid: crystalline
Details on test material:
Identification: Acetamid
Physical Appearance: Colorless crystal substance
Composition: Pure substance >99%

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Test system: Mice, CBA/CaOlaHsd
Rationale: Recognised as the recommended test system.
Source: Harlan Laboratories B.V.Postbus 6174,5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age: Pre-test: 9 - 10 weeks (beginning of treatment)
Main study: 9 - 11 weeks (beginning of treatment)
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet
(certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity approx. 45-65%, except for few hours on
one day (see deviation)
artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
other: Ethanol/water (7+3, v/v)
Concentration:
The test item in the main study was assayed at 10, 25, and 50% (w/v). The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.
No. of animals per dose:
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Any other information on results incl. tables

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

15

---

---

---

---

---

BG II

57

---

---

---

---

0

1

3260

3224.0

8

403.0

1.00

10

2

3041

3005.0

8

375.6

0.93

25

3

3996

3960.0

8

495.0

1.23

50

4

3502

3466.0

8

433.3

1.08

1    =  Control Group

2-4=  Test Group

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 S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In the study the test item Acetamid formulated in ethanol/water (7+3, v/v) was assessed for its possible skin sensitising potential following OECD Guideline 429.
For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/v).
The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.
In this study Stimulation Indices (S.I.) of 0.93, 1.23, and 1.08 were determined with the test item at concentrations of 10, 25, and 50% (w/v) in ethanol/water (7+3, v/v), respectively.
The test item Acetamid was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible skin sensitising potential of Acetamid, three groups each of four female mice were treated once daily with the test item at concentrations of 10, 25, and 50% (w/v) in ethanol/water (7+3, v/v) by topical application to the dorsum of each ear for three consecutive days. A control group of four mice was treated with the vehicle (ethanol/water (7+3, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, 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 of3H‑methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of3HTdR 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 0.93, 1.23, and 1.08 were determined with the test item at concentrations of 10, 25, and 50% (w/v) in ethanol/water (7+3, v/v). A dose response was not observed. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3. The test item Acetamid was not a skin sensitiser under the test conditions of this study.