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: Study was conducted under GLP according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Allantoin
Batch No.: E 03/09
Purity: 99.6 %
Stability in solvent: For days in water and dimethylsulfoxide at room temperature
Storage: At room temperature
Expiration Date: October 09, 2010

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 25 g
- Housing: single caging
- Diet (e.g. ad libitum): pelleted standard diet (Harlan Laboratories GmbH, 33178 Borchen), ad libidum
- Water (e.g. ad libitum): tap water, (Gemeindewerke, 64380 Rossdorf), 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 will be used for the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 45-85%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 a.m. - 6:00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
5, 10, and 20%
No. of animals per dose:
4
Details on study design:
In order to study a possible allergenic potential of Allantoin, three groups each of four female mice were treated with different concentrations of the test item by topical application at the dorsum of each ear lobe (left and right) on three consecutive days. A control group of four mice was treated with the vehicle only. Five days after the first topical application, the mice were intravenously injected into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed and 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 of 3H-methyl thymidine measured in a beta-scintillation counter.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

Positive control results:
Experiment performed in June 2009, 5, 10, and 25% alpha-Hexylcinnamaldehyde yielded a S.I. of 1.79, 2.09, and 6.84, respectively. The EC3 value calculated was 12.9%

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table below

Any other information on results incl. tables

Test item concentration % (w/v)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

16

---

---

---

---

---

BG II

16

---

---

---

---

---

1

3221

3205

8

400.6

 

5

2

3594

3578

8

447.3

1.12

10

3

3036

3020

8

377.5

0.94

20

4

2506

2490

8

311.3

0.78

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 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.

Earweights

The measured ear weights of all animals treated were after necropsy. A relevant increase in ear weights was not observed.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item Allantoin was not a skin sensitiser under the described conditions.
Executive summary:

The present study (Harlan CCR, 2009) analyses the sensitising potential of Allantoin.

Three groups each of four female mice were treated daily with the test item at concentrations of 5, 10, and 20% (w/v) in dimethylsulfoxide by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (dimethylsulfoxide) 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 of 3H-methyl thymidine measured in a beta scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed. A relevant increase in ear weights was not observed for all concentrations tested.

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 1.12, 0.94, and 0.78 were determined with the test item at concentrations of 5, 10, and 20% in dimethylsulfoxide. The EC3 value could not be calculated, since all obtained SI´s were below 3.