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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 April 2012 - 9 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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:
Acetoacetamide
EC Number:
227-774-4
EC Name:
Acetoacetamide
Cas Number:
5977-14-0
Molecular formula:
C4H7NO2
IUPAC Name:
acetoacetamide
Test material form:
solid
Details on test material:
Stability in solvent: > 72 hours in DMSO and ethanol at room temperature
Storage: At room temperature
Expiration Date: July 2012 (Statement of producer)
Specific details on test material used for the study:
Batch No.: BCBD9710V

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS: mice, CBA/CaOlaHsd
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 10 - 11 weeks
- Weight at study initiation: 17.6 - 21.5 g
- Housing: group caging
- Diet: pelleted standard diet (Harlan Laboratories B.V., 5960 AD Horst, The Netherlands), ad libidum
- Water: tap water, (Gemeindewerke, 64380 Rossdorf, Germany), ad libitum
- Acclimation period: 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.
- Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25, and 50% (w/v)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used, was a 50 % solution in dimethylformamide. Vortexing and warming to 37°C was used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37°C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local skin irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Annex 1).
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
Thus, 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 in the pre-experiment.


MAIN STUDY:

TOPICAL APPLICATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear (left and right) with different test item concentrations of 10, 25, and 50% (w/v) in dimethylformamide. The application volume, 25 µL was spread over the entire dorsal surface (Ø~ 8 mm) of each ear once daily for three consecutive days. A futher group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The ANOVA (Dunnett-test) was conducted on the ear weights to assess whether the difference was statistically significant between test item groups and negative control
(vehicle) group.
However, both biological and statistical significance were considered together.

Results and discussion

Positive control results:
Experiment performed in April 2012 (Harlan study number 1486301) using concentrations of 5, 10, and 25 % alpha-hexyl cinnamic aldehyde in acetone:olive oil (4:1). These concentrations yielded S.I.´s of 0.88, 1.51, and 3.73, respectively.
The EC3 value calculated was 20.1 % (w/v).
The positive control substance alpha-hexyl cinnamic aldehyde was found to be a skin sensitizer under the described conditions, demonstrating the validity of the study.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
0.73
Test group / Remarks:
2
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Remarks:
test item conc. 10%w/v
Key result
Parameter:
SI
Value:
0.84
Test group / Remarks:
3
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Remarks:
test item conc. 0.84%w/v
Key result
Parameter:
SI
Value:
0.63
Test group / Remarks:
4
Remarks on result:
no indication of skin sensitisation based on QSAR/QSPR prediction
Remarks:
test item conc. 50%w/v

Any other information on results incl. tables

Calculation and results of individual data; Vehicle: dimethylformamide

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

34

---

---

---

---

---

BG II

24

---

---

---

---

0

1

2589

2560

8

320.0

1.00

10

2

1893

1864

8

233.0

0.73

25

3

2181

2152

8

269.0

0.84

50

4

1637

1608

8

201.0

0.63

BG =  Background (1 mL 5% trichloroacetic acid) in duplicate

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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test item Acetoacetamid was not a skin sensitiser under the test conditions of this study.
Executive summary:
In this study the test item Acetoacetamid dissolved in dimethylformamide was assessed for its possible skin sensitising potential

For this purpose a local lymph node assay according to OECD TG 429 was performed using test item concentrations of 10, 25, and 50% (w/v).

The highest concentration tested was the highest concentration that could be technically achieved and applied whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment.

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. A statistically significant increase in ear weight (p < 0.05) was

not observed in comparison to the values of the vehicle control group (dimethylformamide).

In this study Stimulation Indices (S.I.) of 0.73, 0.84, and 0.63 were determined with the test item at concentrations of 10, 25, and 50% (w/v) in dimethylformamide, respectively. A dose response was not observed.

The test item Acetoacetamid was not a skin sensitiser under the test conditions of this study.

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