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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 19 JUL 2006 to 21 SEP 2006
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:
2006
Report date:
2006

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(4-nitrophenyl)azo]-3-oxobutyramide
EC Number:
258-221-5
EC Name:
N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-2-[(4-nitrophenyl)azo]-3-oxobutyramide
Cas Number:
52846-56-7
IUPAC Name:
2-[(4-nitrophenyl)diazenyl]-3-oxo-N-(2-oxo-2,3-dihydro-1H-benzimidazol-5-yl)butanamide

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: 7-8 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 (%): 30-88
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:water (7+3)
Concentration:
0, 6.25, 12.5, and 25% (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: ethanol:water (7+3) was selected because the test item suspended best in this vehicle
- 25% (w/v) was the highest technically applicable concentration
- Irritation: no signs of toxicity after single application of test item concentration of 25% (w/v); irritating effects such as redness could not be detected due to yellow staining at concentrations of 6.25, 12.5, and 25% (w/v)
- 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 2.3, 4.31 and 11.21 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 (= 6.7% (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: 6.25% test item: 1.14 12.5% test item: 1.54 25% test item: 1.30
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Lymph nodes of the animals of a dose group were pooled: Background of measurement: 22.04 - 22.77; mean: 22.4 (was taken for calculations) control group: 2606.76 6.25% test item: 2967.76 12.5% test item: 4001.32 25% test item: 3373.62

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 node
b)

S.I.

---

BG I

22.04

---

---

---

---

---

BG II

22.77

---

---

---

---

---

CG 1

2606.76

2584.4

8

323.0

 

6.25

TG 1

2967.76

2945.4

8

368.2

1.14

12.5

TG 2

4001 .32

3978.9

8

497.4

1.54

25

TG 3

3373.62

3351.2

8

418.9

1.30

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 under GLP conditions the test item was not a skin sensitizer.
Executive summary:

In the study the test item suspended in ethanol:deionisedwater (7+3) was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 6.25, 12.5 and 25% (w/v).

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. Due to the colour of the test item redness of the ears could not be examined.

In this study Stimulation Indices (S.I.) of 1.14, 1.54 and 1.30 were determined with the test item at concentrations of 6.25, 12.5 and 25% (w/v) in ethanol:deionised water (7+3), respectively.

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