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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 5 APR 2005 to 13 JUN 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 429)

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide
EC Number:
268-734-6
EC Name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-oxo-2-[[2-(trifluoromethyl)phenyl]azo]butyramide
Cas Number:
68134-22-5
Molecular formula:
C18H14F3N5O3
IUPAC Name:
3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)-2-{[2-(trifluoromethyl)phenyl]diazenyl}butanamide
Test material form:
solid: bulk

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands BV, Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Housing: Individually in Macrolon Type I cages with wire mesh top and granulated sooft wood bedding
- Diet: Pelleted standard diet (provided by Harlan Winkelmann GmbH, Borchen, Germany); ad libitum
- Water: Community tap water from Rossdorf, ad libitum
- Acclimation period: yes, but duration not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
0, 5, 10, and 20% (W/v)
No. of animals per dose:
4 females per dose group
2 females in the pre-test
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: DMSO was selected because in any other vehicle test item concentrations above 20% could not be obtained
- 20% (w/v) was the highest technically applicable concentration
- Irritation: no systemic toxicity after single application of test item concentrations of 2.5, 5, 10 or 20% (w/v); irritating effects could not be assessed due to yellow staining at concentrations of 10 and 20% (w/v), but no ear swelling was observed
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
1: exposure to at least one concentration of the test item resulted in an incorporation of 3H-methyl thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index
2: data are compatible with a conventional dose response, although allowance must be made for either local toxicity or immunological suppression

TREATMENT PREPARATION AND ADMINISTRATION:
- individual preparation of weight volume dilutions using a magnetic stirrer as homogenizer
- test item preparations were made feshly before each dosing occasion
- 25 µl were spread over the entire dorsal surface of each ear lobe once daily for three consecutive days
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
- calculations of mean values and standard deviations for body weight
- the EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c; where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining Iymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the value of 3 on the local lymph node assay dose response Plot.

Results and discussion

Positive control results:
Stimulation indices of 2.29, 3.21 and 8.44 were determined with the positive control item at concentrations of 5%, 10% and 25% (w/v), respectively, in acetone:olive oil, 4:1 (v/v). An EC3 value was calculated (= 8.9% (w/v)).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 5% test item: 1.87 10% test item: 1.79 20% test item: 2.35
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Background of measurement: 16.9 - 28.9; mean: 22.9 (was taken for calculations) control group: 4397.9 5% test item: 8187.8 10% test item: 7868.1 20% test item: 10291.3

Any other information on results incl. tables

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Calculation of the EC3 value was not performed because no test concentration produced a SI of 3 or higher.

Test item
concentration
% (w/v)

Group

Measurement
DPM

Calculation

Result

DPM-BGa)

number of
lymph nodes

DPM per
lymph nodeb)

S.I.

---

BG I

28.9

---

---

---

---

---

BG II

16.9

---

---

---

---

---

CG 1

4397.9

4375.0

8

546.9

 

5

TG 2

8187.8

8164.9

8

1020.6

1.87

10

TG 3

7868.1

7845.2

8

980.7

1.79

20

TG 4

10291.3

10268.4

8

1283.6

2.35

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

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Tested in this study according to OECD test guideline no. 429 and GLP conditions the test item was not a skin sensitizer.
Executive summary:

In the study the test item dissolved in DMSO was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 20 %.

No cases of mortality were observed.No symptoms of systemic findings were observed during the study period. Due to the intense yellow colour of the test item at 10 and 20 % (w/v) local irritation reactions such asear redness could not be detected. No swelling of the ears was observed at any of the animals.

In this study Stimulation Indices (S.I.) of 1.87, 1.79, and 2.35 were determined with the test item at concentrations of 5, 10, and 20 % (w/v) in DMSO, respectively.

The test item was found to be not a skin sensitiser in this assay.