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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:
other: GLP 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:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modified LLNA (IMDS): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discriminate the irritating potential from the sensitizing potential of the test substance.
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorized in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Light yellow, clear liquid; molecular weight: 123 g/mol; content: > 99 %

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win:NMRI
- Age at study initiation: 9 weeks
- Weight at study initiation: 26 - 34 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2
- Humidity (%): 40 - 70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
The stability of the test item in the vehicle was analytically verified for up to 4 days.
Concentration:
0, 2, 10, 50 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation and the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25µl/ear. The used concentrations were based on the experiences with the test system and the toxic properties of the test substance.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiiological saline (PBS).
Investigations:
- weight of lymph nodes
- cell counts in lymph nodes
- stimulation index is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling
- ear weight
- body weights
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5% . Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99% by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differentes in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:
Alpha hexyl cinnamic aldehyde, checked in regular intervals, shows a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Compared to vehicle-treated animals, none of the parameters measured in the test substance groups (nitrobenzene concentrations of 2 %, 10 % and 50 % in acetone/olive oil), i.e. cell counts, weights of the draining lymph nodes, ear weights and ear swelling reached or exceeded the 'positive levels' defined for this assay. The cell count indices were determined with 0.96, 0.82 and 0.86. The critical 'positive level' for this parameter is defined with 1.4.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

Any other information on results incl. tables

The "positive level" of ear swelling which is 2 x 10-2 mm increase, i.e. about 10% of the control values, has not been reached or exceeded in any dose group. A slight statistically significant increase in ear swelling was determined for the 2% group on day 4. This increase is within the normal range of variance for this parameter, which means that it is only of statistical significance because the mathematical conditions were favorable. No substance specific effects were determined for ear weights either.

It has to be clarified that the "positive levels" mentioned above are exclusively defined for the NMRI outbred mice used for this study. Such positive limits have to be calculated for each strain of mice individually.

The body weights of the animals were not affected by any treatment.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Executive summary:

Nitrobenzene showed no sensitizing potential in the modified Local Lymph Node Assay (IMDS) in female NMRI mice after dermal application of up to and including a 50 % concentration. Additionally, no indication for a non-specific (irritant) activation was detected. Therefore, the concentration of 50 % turned out to be the NOEL for the parameters investigated in this study with respect to skin sensitization.