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Description of key information

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
Study period:
April 2000
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
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: Harlan Winkelmann GmbH, Borchen, Germany
- Strain: Hsd Win:NMRI (SPF)
- Housing: in groups up to 6 animals
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): about 50
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
other: DAE 433: mixture of dimethylacetamide (40%), acetone (30%) and ethanol (30%)
Concentration:
0, 1, 10, 50 %
No. of animals per dose:
6
Details on study design:
TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation or the vehicle were applied epicutaneously onto the dorsal part of both ears of the animals. This  treatment was repeated on three consecutive days (d0, d1 and d2). 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 (d3). 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)
Positive control substance(s):
not specified
Statistics:
When it was statistically reasonable, the values from treated groups were compared with those from the control group by the Mann-Whitney or the Wilcoxon signigicance test (U-test) at significance levels of 5 and 1% (one-tailed for LLNA/IMDS or PNLA (larger)). Outlying values in the LN/ear weights or LN cell counts 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.
Parameter:
SI
Remarks:
cell count in draining lymph nodes
Value:
1.03
Test group / Remarks:
1%
Parameter:
SI
Remarks:
cell count in draining lymph nodes
Value:
0.75
Test group / Remarks:
10%
Parameter:
SI
Remarks:
cell count in draining lymph nodes
Value:
0.98
Test group / Remarks:
50%
Parameter:
SI
Remarks:
weight of draining lymph nodes
Value:
1.09
Test group / Remarks:
1%
Parameter:
SI
Remarks:
weight of draining lymph nodes
Value:
0.91
Test group / Remarks:
10%
Parameter:
SI
Remarks:
weight of draining lymph nodes
Value:
0.94
Test group / Remarks:
50%

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

Parameter investigated

Vehicle

control

Dose  1%

Dose 10 %

Dose  50%

Stimulation index:

weight of draining lymph nodes

1.00

1.09

0.91

0.94

Stimulation index:

cell count in draining lymph nodes

1.00

1.03

0.75

0.98

Ear swelling in 0.01 mm on day 3 (index)

20.92

(1.00)

20.33

(0.97)

20.08

(0.96)

19.75

(0.94)

Ear weight in mg / 8 mm diameter punch on day 3 (index)

13.82

(1.00)

13.68

(0.99)

13.34

(0.97)

13.63

(0.99)

The mice did not show any significant dose-dependent increase in the stimulation indices for the weight or cell counts as well as for ear swelling or ear weights and statistical analysis revealed no significant effect for the test item. In addition, no values were found exceeding the "positive levels" which are 1.25 for cell count index and 2x10-2 mm increase for ear swelling, respectively.

Interpretation of results:
GHS criteria not met
Executive summary:

Ethoxybenzonitril was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429. Concentrations of 0 (vehicle control), 1, 10 and 50 % formulated in DAE 433 were tested. The results show that the test item has no sensitizing potential in mice after dermal application. A significant difference compared to vehicle treated animals regarding the weight or cell counts of the draining lymph nodes as well as ear swelling was not reached in any case.

 

Thus, no hint for a substance specific or non-specific activation of the cells of the immune system via dermal application was found by the method used.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Ethoxybenzonitril was investigated in the modified local lymph node assay (LLNA-IMDS) on female mice according to OECD TG 429 (Vohr, 2000). Concentrations of 0 (vehicle control), 1, 10 and 50 % formulated in DAE 433 were tested. The results show that the test item has no sensitizing potential in mice after dermal application. A significant difference compared to vehicle treated animals regarding the weight or cell counts of the draining lymph nodes as well as ear swelling was not reached in any case.

 

Thus, no hint for a substance specific or non-specific activation of the cells of the immune system via dermal application was found by the method used.


Migrated from Short description of key information:
Ethoxybenzonitril has no skin sensitizing potential.

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

Based on the study results a classification according to Directive 67/548/EEC or Regulation (EC) No. 1272/2008 (CLP) is not warranted.