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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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
Chemical structure
Reference substance name:
3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]
EC Number:
226-970-7
EC Name:
3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-(3-chloro-o-tolyl)benzamide]
Cas Number:
5580-57-4
Molecular formula:
C43H35Cl5N8O6
IUPAC Name:
3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis[4-chloro-N-(3-chloro-2-methylphenyl)benzamide]
Test material form:
solid: particulate/powder
Details on test material:
Pigment Yellow 93
Specific details on test material used for the study:
- Lot/batch No.:7218
- Expiration date of the lot/batch: 02/ 2023
- Stability under test conditions: stable
- Storage condition of test material: The substance was indicated to be stored in the airtight delivered plastic container in the darkness at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic, ReH CZ 21760118
- Age at study initiation: 8 to 10 weeks (at start of dosing)
- Weight at study initiation: 17,3 to 21,6 g (at start of dosing)
- Housing: in groups of maximum six
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

IN-LIFE DATES: From: 3.11.2008 To: 5.11.2008

Study design: in vivo (LLNA)

Vehicle:
other: DAE 433 - mixture of40% dimethylacetamide, 30% acetone and 30% ethanol
Concentration:
0.1, 1 and 10%
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: The test item is a pigment and insoluble in organic or inorganic solvents. A suspension of up to 10% could be tested.
- Irritation: Not irritating
- Lymph node proliferation response: No data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: non-radiolabelled LLNA
- Criteria used to consider a positive response: The results of the LLNA were evaluated according to the following criteria.
The following thresholds were determined by Ulrich (2007) from analysis of historical data:
Ear weight index: 1.05
LN weight: 1.2
LN cell count: 1.3

1. Values which exceed these thresholds were considered positive
- when a statistically significant increase in one of the parameters occurs and a clear
concentration-dependence can be derived
- or with no statistical significance, but a clear concentration-dependence.
2. Values which are below these thresholds were considered positive
- when a statistical significant occurs in one of the parameters together with a clear
concentration dependence.

TREATMENT PREPARATION AND ADMINISTRATION: Before start of application the suspension was mixed for 5 minutes with a magnetic stirrer and then were still mixed during application.
Positive control substance(s):
other: Dinitrochlorobenzene
Statistics:
For statistical calculations the software Statgraphic® Centurion (version XV, USA) was used. At first the global comparison of all three values of the concentration groups with vehicle control was performed by applying the non-parametric Kruskal-Wallis test, and then the non-parametric two-group Mann-Whitney rank test (probability level 0.05) was applied to all two-group comparisons.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: LN cell count index
Value:
1
Test group / Remarks:
Negative control
Parameter:
other: LN cell count index
Value:
4.06
Test group / Remarks:
Positive control
Key result
Parameter:
other: LN cell count index
Value:
0.84
Test group / Remarks:
0.1% test substance
Key result
Parameter:
other: LN cell count index
Value:
0.84
Test group / Remarks:
1% test substance
Key result
Parameter:
other: LN cell count index
Value:
1.04
Test group / Remarks:
10% test substance

Any other information on results incl. tables

Mean local lymph node weight (mg)

positive control: 14,87

Negative contro: 5,68

Test substance 0.1% : 5,77

Test substance 1%: 5,68

Test substance 10%: 5,45

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
at the given experimental conditions the test substance elicited negative result in LLNA test. Based on these results the test substance does not have to be classified as the substance, which may cause sensitisation by skin contact.
Executive summary:

In this study the contact allergenic potential of the test item was evaluated with non radioactive measuring of cell proliferation after topical application to female BALB/c mice. Six mice per group were exposed by test and control substances on the dorsum of both ears once a day during 3 consecutive days. Draining lymph nodes were taken off at 24 hours after the last application. Concentrations: positive control DNCB (dinitrochlorobenzene): 0.5% (w/v) and test item: 10%, 1%, 0.1% (w/v) in DAE 433. Endpoints: ear weight, auricular (ear-draining) lymph node weights and cell counts = lymph node (LN) hyperplasia.


The animals exposed to the test substance at the highest concentration showed skin reactions (congestion of vessels) throughout the experiment. There were no clinical observations attributable to the treatment with test substance at the middle and lower dose level. There was no difference in body weight increment of all groups in comparison to the vehicle control. The positive control substance DNCB elicited a reaction pattern with statistically significant increase in ear weight and LN hyperplasia, which was in congruence with his expected mode of action as a contact allergen. The test substance showed a tendency to increase of ear weight in the highest dose level but without lymph node hyperplasia. Residues of the test substance on the ears caused this increased weight. Comparison of values between treated groups and control group revealed that the test substance did not cause statistically significant increase in LN cell count or in LN weight. Also index of LN weight and LN cell count was not exceeded in any dose level.


In conclusion, at the given experimental conditions the test substance elicited negative result in LLNA test. Based on these results the test substance does not have to be classified as the substance, which may cause sensitisation by skin contact.