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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:
2013-01-28 to 2013-02-28
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)
Version / remarks:
2010
Deviations:
yes
Remarks:
Measurement of the cell proliferation by cell counting 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.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
1998
Qualifier:
according to
Guideline:
other: Note for Guidance on Non-Clinical Tolerance Testing of Medicinal Products, CPMP/SWP/2145/00, adopted March 1, 2001
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 the Note of
Guidance SWP/ 2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: 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 Laboratories Germany GmbH, 97633 Sulzfeld, Germany
- Sex: female
- Age: 62 days
- Weight at study initiation: 28 - 33 g
- Housing: single
- Diet: ad libitum, ssniff R/M-H V 1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water: ad libitum, tap water
- Acclimatisation period: at least 5 days
- Controls: 6 female mice, treated with vehicle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3 °C
- Humidity (%): 55 % +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle
- Air exchange: 12 to 18 times per hour
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
vehicle, 25 % w/w, 50% w/w, 100% test item
No. of animals per dose:
6 female mice
Details on study design:
RANGE FINDING TESTS:
- 3 animals/test item to examine irritating potential and handling/application to select the appropriate concentrations. Two concentrations of 25%
and 50% of the test item in acetone / olive oil (3 + 1, v/v), w/w, and the undiluted test item were examined.
TREATMENT PREPARATION AND ADMINISTRATION:
- Three concentrations of 25% and 50 % of test item in acetone / olive oil (3 + 1, v/v), w/w and the undiluted test item were
examined.
- Vehicle: olive oil (3 + 1 v/v)
- Weight of each animal, clinical observations and ear swelling measurements at the helical edge of both ears using an Oditest micrometer are
recorded
Open application of 25 µl
- vehicle alone
- test item in 3 concentrations
- positive control onto the dorsal part of both ears of the animals.
This treatment was repeated on three consecutive days (d1, d2 and d3).
24 h after last application (day 4), ear swelling measurements are carried out at the helical edge of both ears using an Oditest micrometer
Immediatly after measurement animals were sacrificed under ether anaesthesia.
The appropriate organs were then removed.
Lymphatic organs (the auricular lymph nodes) were then stored on ice in PBS /0.5% BSA.
Investigations:
- ear swelling
- ear weight (Punch biopsies of 8 mm of the apical area)
- 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.
Animals were observed once daily for clinical signs of local systemic irritation at the application site or of systemic toxity. Body weight were recorded on day 1 and day 4.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The so-called stimulation (or LLN-) index to establish the sensitising potential is calculated by dividing the average absolute lymph node weight or
lymph node cell counts per group of the test item treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index
for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive (sensitising properties). For lymph node
weight a significance at p ≤ 0.01 is considered positive, however, an increase in lymph node weight is an indication for possible irritating properties
not sensitising properties.
In addition, the average ear weights per group and the average ear thickness per group will be compared to the vehicle control group as an indication for possible irritating properties.
Parameter:
SI
Remarks on result:
other: see below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: modified LLNA; measurement of cell counts instead of radioactive labeling

Main study results: stimulation indices (SI):

Parameter

Group 1, negative control

Group 2,

25%

Group 3, 50%

Group 4, 100%

Group 5,

positive control

Lymph node cell count

1.000

1.089

1.013

1.140

1.419

Lymph node weight

1.000

1.000

1.020

1.078

1.412

Ear weight

1.000

1.054

1.048

1.048

1.151

Difference of

ear thickness

1.000

1.000

1.017

1.050

1.151

____ significantly different from control at p ≤ 0.01

Conclusions:
Under the present test conditions, the test item at concentrations of 25% or 50% (w/w) in acetone/olive oil
(3+1 v/v) or the undiluted test item did not reveal any sensitising properties inthe modified local lymph node assay.
Executive summary:

The purpose of this study was to determine the sensitising potential of the test substance in the modified local lymph node assay in mice.

The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form oflymph node cell counts and lymph node weightsif justification and appropriate scientific support exist showing the validity of this method.

The alternative method used for the study employing thelymph node weightandlymph nodecell count to assessproliferationhas been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b.This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.

In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the vehicle treated ones.

Values above 1.4 (lymph nodecell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of1,6-Di(trimethoxysilylpropylcarbamoyloxy)hexane(25% and 50%, suspended in acetone/olive oil (3+1, v/v))(w/w) or the undiluted test item (100%) were tested in six female NMRI mice per group and compared to a vehicle control group.

In addition, a positive control group(25% solution v/v ofa-hexyl cinnamic aldehyde inacetone/olive oil (3+1, v/v)) was employed.

 

Stimulation indices (SI):

Parameter

Group 1, negative control

Group 2,

25%

Group 3, 50%

Group 4, 100%

Group 5,

positive control

Lymph node cell count

1.000

1.089

1.013

1.140

1.419

Lymph node weight

1.000

1.000

1.020

1.078

1.412

Ear weight

1.000

1.054

1.048

1.048

1.151

Difference of

ear thickness

1.000

1.000

1.017

1.050

1.151

____ significantly different from control at p ≤ 0.01

Treatment with the test substance at concentrations of 25%, 50% or 100% did not reveal statistical significantly increased values forlymph nodecell count. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4. Hence, the test item is classified as not sensitising.

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted.

The positive control group caused the expected increases in lymph node cell countand lymph node weight(statistically significant at p ≤ 0.01).Therefore, the study can be regarded as valid.

No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, the test substance at concentrations of 25% or 50% (w/w) in acetone/olive oil (3+1, v/v) or the undiluted test item did not reveal any sensitising properties in the local lymph node assay.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
Under the present test conditions, the test substance at concentrations of 25% or 50% (w/w) in acetone/olive oil
(3+1 v/v) or the undiluted test item did not reveal any sensitising properties inthe modified local lymph node assay.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:
Migrated from Short description of key information:
Assessment of the test substance concerning respiratory sensitisation is not possible because no data are available for this toxicological endpoint.

Justification for classification or non-classification

Based on the results of the modified LLNA and according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008 the test item is not classified as sensitizing to skin.