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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:
2014-02-19 to 2014-04-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-[(2-methyl-1,3-phenylene)diimino]bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
EC Number:
225-744-5
EC Name:
3,3'-[(2-methyl-1,3-phenylene)diimino]bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Cas Number:
5045-40-9
Molecular formula:
C23H8Cl8N4O2
IUPAC Name:
3,3'-[(2-methyl-1,3-phenylene)diimino]bis(4,5,6,7-tetrachloro-1H-isoindol-1-one)
Constituent 2
Chemical structure
Reference substance name:
monomethoxy-heptachloro derivative of 3,3'-[(2-methyl-1,3-phenylene)diimino]bis[2,3-dihydro-1H-isoindol-1-one]
Molecular formula:
C24H11Cl7N4O3
IUPAC Name:
monomethoxy-heptachloro derivative of 3,3'-[(2-methyl-1,3-phenylene)diimino]bis[2,3-dihydro-1H-isoindol-1-one]
Test material form:
solid
Details on test material:
- Analytical purity: 100 % UVCB
- Storage condition of test material: At room temperature, protected from light
- Physical state/appearance: yellow solid

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V. Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: first pretest: 9-10 weeks, second pretest: 8-9 weeks, main study: 8-9 weeks
- Weight at study initiation: mean: 20.1 g (18.5 - 22.4 g)
- Housing: group
- Diet: 2018C Teklad Global 18 % protein rodent diet, ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity (%): 45-65
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
dimethyl sulphoxide
Concentration:
1, 2 and 5 % in DMSO
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Irritation: no visible erythema, redness was not determined due to inherent colour of test item

MAIN STUDY
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.
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, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as a statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05).

Results and discussion

Positive control results:
The sensitivity and reliability of the experimental technique employed was assessed by use of α-hexyl cinnamaldehyde dissolved in acetone/olive oil (4+1, v/v) (compound listed in OECD 429 Guideline) which is known to have skin sensitisation properties in mice. The periodic positive control experiment was performed using CBA/CaOlaHsd mice in October 2013.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
0.83
Test group / Remarks:
1% test item concentration
Parameter:
SI
Value:
1.03
Test group / Remarks:
2% test item concentration
Parameter:
SI
Value:
0.96
Test group / Remarks:
5% test item concentration

Any other information on results incl. tables

A statistically significant or biologically relevant increase in DPM values, lymph node weights and lymph node cell counts was not observed in any treated group in comparison to the vehicle control group.


 


Table 1: DPM and S.I.







































 


Test item concentration



Group Calculation



Mean DPM per animal (2 lymph nodes)*



 SD



S.I.



Vehicle Control Group (DMSO)



2198.2



1226.3



1.00



1% test substance



1828.8



486.4



0.83



2% test substance



2265.8



783.4



1.03



5% test substance



2119.4



258.0



0.96



 


* Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals).


 


 


The EC3 value could not be calculated, since all S.I.´s were below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met