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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:
from september 7, 2005 to september 21, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: LLNA test, guideline test with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The age of the animals was 6 to 7 weeks, instead of 8 to 12 weeks and the relative humidity in the animal room was between approximately 30 - 80 %, these deviations to the study plan, however, did not affect the validity of the study,
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
Animals:
Test system:Mice, CBA/CaOlaHsd
Rationale:Recognized as the recommended test system
Source:Harlan Netherlands B.V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
Number of animals for the pre-test (non-GLP): 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant) 3
Number of test groups: 3
Number of control (vehicle): 1
Age: 6-7 weeks(beginning of acclimatization)
Identification:Single caging. The animals will be distributed into the test groups at random and identified by cage number.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness will be used for the study.
Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen)
Bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
Feed: pelleted standard diet, ad libitum(Harlan Winkelmann GmbH, D-33178 Borchen)
Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
Environment: temperature 22±3°C relative humidity 30-80% artificial light 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
0 (control), 12.5, 25 and 50%
No. of animals per dose:
4
Details on study design:
In order to study a possible allergenic potential of test article, 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 p-scintiation 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:
Test item concentration
% (w/v) Group Measurement
DPM Calculation Result
DPM-BG a) number of lymph nodes DPM per lymph node b) S.I.
… BG I 11.1 --- … … …
… BG II 4.8 --- … … …
… CG 1 5836.1 5828.2 8 728.5
5 TG 2 6162.5 6154.6 8 769.3 1.06
10 TG 3 10534.7 10526.8 8 1315.8 1.81
25 TG 4 23323.7 23315.8 8 2914.5 4.00
BG = Background (1 ml 5% trichloroacetic acid) in duplicate CG = Control Group TG = 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 10.0 (a) 1.81 (b)
Group 3 25.0 (c) 4.0 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 18.2 % (w/v)
EC3 = Estimated concentration for a S.I. of 3.
a,b,c,d = Co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see table 1 in the field for additional information of results.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table 1 in the field for additional information of results.

Any other information on results incl. tables

 Calculation and Results of Individual Data

Test item concentration

% (w/v)

Group

Measurement

DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

BG I

23.61

BG II

24.94

CG 1

4310.73

4286.5

 8

535.8

 

12.5

TG 2

7046.78

7022.5

 8

877.8

1.64

25

TG 3

5740.50

5716.2

 8

714.5

1.33

50

TG 4

6927.91

6903.6

 8

863.0

1.61

BG = Background (1 ml 5% trichloroacetic acid) in duplicate CG = Control Group TG = 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 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 with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, test article was shown to be not a skin sensitizer in this murine local lymph node assay.
Executive summary:

This study was performed in compliance with Good Laboratory Practice Regulations and OECD guideline 429: Skin Sensitisation. Local Lymph Node Assay (adopted 24 April 2002). Test article dissolved in DMF was assessed for its possible contact allergenic potential in the murine local lymph node assay (LLNA) in CBA/CaOlaHsd mice.

The ears of each 4 female mice were topically treated with the test item in concentrations of 0 (control), 12.5, 25 and 50% for three consecutive days. Five days after the first topical application the mice were injected intravenously with 3H-methyl thymidine. Approximately five hours thereafter, the mice were sacrificed, the draining lymph nodes excised and pooled. Single cell suspensions of lymph node cells were prepared and the incorporation of 3H-methyl thymidine was measured in a p-scintillation counter.

The animals did not show any local findings or clinical signs of systemic toxicity during the study and no mortality was observed.

The Stimulation Indices (S.I.) of 1.64, 1.33 and 1.61 were determined with the test item at concentrations of 12.5, 25 and 50% (w/v) in DMF, respectively, and thus, were clearly below the limit criterium of a S.I. of 3.