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Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
- modification: in addition, measurements of ear swelling and ear weight were done to discriminate the irritating potential from the sensitizing potential of the test substance (Integrated Model for the Differentiation of Skin reactions (IMDS))
Principles of method if other than guideline:
Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised 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)
Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain: Crl:NMRI BR
- Age at study initiation: 7 weeks
- Weight at study initiation: 27 - 35 g
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days

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

Vehicle:
methyl ethyl ketone
Concentration:
0, 10 %, 30 % and 100 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation, the positive control or 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 physiological saline (PBS).
Investigations:
- weight of draining lymph nodes (given as weight index compared to vehicle controls)
- cell counts in draining lymph nodes (given as cell count index compared to vehicle controls)
Stimulation indices were calculated by dividing the absolute weight or number of cell counts of the substance treated lymph nodes by the vehicle treated ones.
- ear swelling (given in 0.01 mm and as index)
- ear weight (given in mg/8 mm diameter punch and as index)
- 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.
Positive control results:
Alpha hexyl cinnamic aldehyde showed a clear sensitizing potential in the local lymph node assay (IMDS).
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Compared to vehicle treated animals there was an increase regarding the the cell count in the high dose group (index of 1.53 %) which was of statistical significance. The "positive level" of index 1.4 for the cell counts, has been exceeded in this dose group. The "positive level" of ear swelling has not been reached or exceeded in any dose group. No substance specific effects were determined for ear weight either. The results show  that M 530 has a weak sensitising potential in mice after dermal application.  The EC 1.4 value calculated for this test item is 80.22 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a weak skin sensitiser.

Table 1: Summary of the LLNA/IMDS results with M 530 (means of 6 animals per group)


Parameter investigated

Vehicle

control

Dose 

  10%

Dose

30 %

Dose

100 %

Positive

control

Stimulation index:

weight of draining lymph nodes

1.00

0.97

1.14

1.30

1.56 *

Stimulation index:

cell count in draining lymph nodes

1.00

1.01

1.07

1.53*

1.77 *

Ear swelling in 0.01 mm on day 4 (index)

17.75

(1.00)

17.42

(0.98)

 17.92

(1.01)

 18.50

(1.04)

22.50*

(1.27)

Ear weight in mg / 8 mm diameter punch on day 4 (index)

12.61

(1.00)

11.64

(0.92)

 11.88

(0.94)

 13.01

(1.03)

15.98 *

(1.27)

 

* statistically significant increase

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

Interpretation of results:
sensitising
Remarks:
Migrated information
Executive summary:

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 10 %, 30 % and 100 % formulated in dimethylformamide were tested. Compared to vehicle treated animals there was a clear increase regarding the cell counts (index of 1.53 %), which is statistical significant. The "positive level" of index 1.4 for the cell counts, has been exceeded in this dose group. The EC 1.4 value calculated for this test item is 80.22 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a weak skin sensitiser.

In summary, these results show that the test item has a weak sensitising potential in mice after dermal application of a 100 % concentration. The concentration of 30 % turned out to be the NOEL for the parameters investigated in this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test item was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 10 %, 30 % and 100 % formulated in dimethylformamide were tested. Compared to vehicle treated animals there was a clear increase regarding the cell counts (index of 1.53 %), which is statistical significant. The "positive level" of index 1.4 for the cell counts, has been exceeded in this dose group. The EC 1.4 value calculated for this test item is 80.22 %. In accordance with the classification proposed in the Technical Report No. 78 of the ECETOC (2003) this value corresponds to a weak skin sensitiser.

In summary, these results show that the test item has a weak sensitising potential in mice after dermal application of a 100 % concentration. The concentration of 30 % turned out to be the NOEL for the parameters investigated in this study.


Justification for selection of skin sensitisation endpoint:
Only one study available

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation

The EC1.4-value calculated is 80.22 % for this test item in the Mouse Local Lymph Node Assay.

According to EU-Directive 67/548/EEC, Annex VI the test item shall be classified as sensitising.

According to Regulation (EC) No 286/2011 of 10 March 2011 amending Regulation (EC) No 1272/2008 the test item shall be allocated to Sub-category 1B.

Respiratory sensitisation:

no data available