Registration Dossier

Diss Factsheets

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
DEC 2004 to 25 JAN 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 429) performed under GLP

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:
yes
Remarks:
due to technical reasons only one lymph node could be analysed for one animal
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-[(2,5-dichlorophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxynaphthalene-2-carboxamide
EC Number:
230-258-1
EC Name:
4-[(2,5-dichlorophenyl)azo]-N-(2,3-dihydro-2-oxo-1H-benzimidazol-5-yl)-3-hydroxynaphthalene-2-carboxamide
Cas Number:
6992-11-6
Molecular formula:
C24H15Cl2N5O3
IUPAC Name:
4-[(1E)-2-(2,5-dichlorophenyl)diazen-1-yl]-3-hydroxy-N-(2-oxo-2,3-dihydro-1H-1,3-benzodiazol-5-yl)naphthalene-2-carboxamide
Test material form:
solid: nanoform, no surface treatment

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:
acetone/olive oil (4:1 v/v)
Concentration:
0, 3.75, 7.5, and 15 % (w/v)
No. of animals per dose:
5 females per dose group
2 females in the pre-test
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: acetone/olive oil (4:1) was selected as solvent because it was the most suitable solvent
- 15% (w/v) was the highest technically applicable concentration
- Irritation: no signs of systemic toxicity after single application of test item concentration of 15% (w/v). Due to the intense brown colour of the test item local irritation reactions such as ear redness could not be detected. No swelling of the ears 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
- A statistical analysis was conducted for assessment of the dose-response relationship, and 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.
- The Dunnett-test (ANOVA) was conducted to assess whether the difference is statistically significant between test item groups and negative control (vehicle) group. Statistical significance was at the five per cent level (p < 0.05).

Results and discussion

Positive control results:
Stimulation indices of 2.0, 3.0 and 4.9 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 (= 9.9% (w/v)).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1
Test group / Remarks:
CG 1_5
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79
Parameter:
SI
Value:
1.35
Test group / Remarks:
TG 2_5
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79
Parameter:
SI
Value:
0.73
Test group / Remarks:
TG 3_5
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79
Parameter:
SI
Value:
0.79
Test group / Remarks:
TG 4_5
Remarks on result:
other: Stimulation indices (SI) were all below 3. The following SI were calculated: 3.75% test item: 1.35 7.5% test item: 0.73 15% test item: 0.79

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

Measurement
DPM

Calculation

Result

Calculation

DPM

per lymph node

b)

Result

% (w/v)

Group

DPM-BG
per animal
(2 lymph nodes)
a)

S.I,*

number
of
lymph nodes

S.I.

---

BG I

2.9

---

---

---

---

----

---

BG II

3.7

---

---

---

---

---

---

CG 1_1

1905.2

1901.9

---

---

---

---

---

CG 1_2

1269.1

1265.8

---

---

---

---

---

CG 1_3

1384.1

1380.8

---

---

---

---

---

CG 1_4

1522.3

1519.0

---

---

---

---

---

CG 1_5

1667.7

1664.4

---

10

773.2

1.00

3.75

TG 2_1

1466.3

1463.0

0.9

---

---

---

3.75

TG 2_2

2294.1

2290.8

1.5

---

---

---

3.75

TG 2_3

2562.7

2559.4

1.7

---

---

---

3.75

TG 2_4

1941.8

1938.5

1.3

---

---

---

3.75

TG 2_5

1112.5

1109.2c)

0.7

9

1040.1

1.35

7.5

TG 3_1

823.7

820.4

0.5

---

---

---

7.5

TG 3_2

1316.3

1313.0

0.8

---

---

---

7.5

TG 3_3

1252.3

1249.0

0.8

---

---

---

7.5

TG 3_4

984.3

981.0

0.6

--

---

---

7.5

TG 3_5

1305.8

1302.5

0.8

10

566.6

0.73s

15.0

TG 4_1

1113.7

1110.4

0.7

---

---

---

15.0

TG 4_2

898.2

894.9

0.6

---

---

---

15.0

TG 4_3

1420.1

1416.8

0.9

---

---

---

15.0

TG 4_4

1352.7

1349.4

0.9

---

---

---

15.0

TG 4_5

1313.8

1310.5

0.8

10

608.2

0.79

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

CG = Control Group

TG = Test Group

5.1 = Stimulation Index

* = mean net DPM value per lymph node of the mouse divided by the mean net DPM value of the vehicle control group

a)        =The mean of the Background value (BGI and BGH) was subtracted from the value.

b)                  DPM/node was determined by dividing the sum of the measured values from all lymph nodes within a group by the number of lymph nodes taken from that group

c)                  DPM value of one lymph node

s = Mean DPM value for the group was according to the Dunnett test significantly lower than corresponding control value. The p value for the analysis was 0.038. This is, however,irrelevant, since the significant value (0.73) is below the control value and not above.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Tested in this study according to OECD test guideline no. 429 and GLP conditions the test item was not a skin sensitiser.
Executive summary:

In the study the test item dissolved in acetone:olive oil, 4:1 (v/v)was assessed for its possible contact allergenic potential.

For this purpose a local lymph node assay was performed using test item concentrations of 0, 3.75, 7.5 and 15 % (w/v).

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.l..) of 1.35, 0.73 and 0.79 were determined with the test item at concentrations of 3.75, 7.5 and 15 % (w/v) in acetone:olive oil, 4:1 (v/v), respectively,

The test item was not a skin sensitiser in this assay