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Toxicological information

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:
01 - 30 Jun 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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:
yes
Remarks:
no radioactive labelling was used to measure cell proliferation
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2012
Deviations:
yes
Remarks:
no radioactive labelling was used to measure cell proliferation
Principles of method if other than guideline:
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation. The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. 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.

References:
Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a)

Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b)
GLP compliance:
yes (incl. QA statement)
Remarks:
FREIE UND HANSESTADT HAMBURG, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
EC Number:
286-056-9
EC Name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol
Cas Number:
85186-72-7
IUPAC Name:
Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

In vivo test system

Test animals

Species:
mouse
Strain:
NMRI
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 62 days (on test day 1)
- Weight at study initiation: 29 - 34 g (test day 1)
- Housing: animals were kept individually in in MAKROLON cages (type III) supplemented with granulated textured wood as bedding material (Granulat A2, J. Brandenburg, Goldenstedt, Germany)
- Diet: commercial diet ssniff R/M-H V1534 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50, 75 and 100%
No. of animals per dose:
6
Details on study design:
PRE-SCREEN TESTS
A preliminary experiment was carried out in 4 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Four concentrations of 25%, 50% and 75% dissolved in acetone / olive oil (4:1, v/v) and the undiluted test item were examined. Doses were selected according to OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% etc. The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. Both ears of each mouse were observed for erythema and scored using following pattern: 0 (no erythema), 1 (very slight erythema), 2 (well-defined erythema) and 4 (moderate to severe erythema).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Alternative endpoints of the murine local lymph node (measurement of lymph node weight and lymph node cell count, ear weight/thickness measurement to detect skin irritation potential)
- Criteria used to consider a positive response: 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 node cell 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).

TREATMENT PREPARATION AND ADMINISTRATION
The test item was used undiluted or diluted in acetone / olive oil (4:1, v/v). The vehicle acetone / olive oil (4:1, v/v) was used as negative reference item. The test item solution was administered to the dorsum of both animals´ ears at an application volume of 25 µL/ear. Five groups of 6 female animals each were examined. The concentrations (50, 75 and 100%) were chosen based on a preliminary experiment. The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation. The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European interlaboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. 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. The experimental schedule of the assay was as follows:
- Day 1: The weight of each animal was individually identified and recorded. The weights and any clinical observation were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer. Open application of 25 μL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3: The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application): Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer. The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised. Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance. Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.
Positive control substance(s):
other: 20% solution (v/v) of α-hexyl cinnamic aldehyde in acetone / olive oil (4:1, v/v)
Statistics:
For lymph node weight significance at p ≤0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship for the lymph node weight in order to determine a possible sensitising potential was examined by linear regression analysis employing PEARSON's correlation coefficient. U-test was performed for cell count, too. Outliers were determined according to the Nalimov test. Hence, in addition, the acute inflammatory skin reaction (irritating potential) was 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 employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for the ear weight stimulation index was set at 1.1.

Results and discussion

Positive control results:
The positive control was dissolved in acetone / olive oil (4:1, v/v). Positive controls were used to demonstrate appropriate performance of the assay and competence of the laboratory to successfully conduct the assay. An index for the lymph node cell count above 1.4 is considered positive. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤0.01). Therefore, the study can be regarded as valid.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.073
Test group / Remarks:
50% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.062
Test group / Remarks:
75% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.091
Test group / Remarks:
100% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.526
Test group / Remarks:
Positive control
Key result
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.137
Test group / Remarks:
50% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node weight
Test group / Remarks:
75% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.176
Test group / Remarks:
100% test item
Remarks on result:
other: no indication of skin sensitisation
Key result
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.471
Test group / Remarks:
Positive control
Key result
Parameter:
SI
Remarks:
Ear weight
Value:
0.98
Test group / Remarks:
50% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear weight
Value:
1.005
Test group / Remarks:
75% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear weight
Value:
1.061
Test group / Remarks:
100% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear weight
Value:
1.097
Test group / Remarks:
Positive control
Key result
Parameter:
SI
Remarks:
Ear thickness
Value:
0.996
Test group / Remarks:
50% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear thickness
Value:
1.008
Test group / Remarks:
75% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear thickness
Value:
0.988
Test group / Remarks:
100% test item
Remarks on result:
other: no indication of skin irritation
Key result
Parameter:
SI
Remarks:
Ear thickness
Value:
1.118
Test group / Remarks:
Positive control
Cellular proliferation data / Observations:
PRELIMINARY EXPERIMENT
In a preliminary experiment, concentrations of 25%, 50%, 75%, and 100% of the test material employing 1 animal per concentration, were examined. No systemic toxicity or excessive local skin irritation were observed in this preliminary experiment at concentrations of 25%, 50%, 75% and 100%.

MAIN STUDY
In the main study treatment with the test material at concentrations of 50%, 75% or 100% did not reveal any statistical significantly increased values for the lymph node cell count. The stimulation indices calculated for the lymph node cell count did not exceed the threshold level of 1.4. In addition, no statistical significantly increase in the lymph node weight was observed. Hence, the test item is classified as not sensitising to the skin. The threshold level of 1.1 for the ear weight stimulation index was not exceeded and no increase of ear thickness was observed, thus, no irritating properties were noted.

CLINICAL OBSERVATIONS
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS
The animal body weight was not affected by the treatment.

Any other information on results incl. tables

Table 1: Summary of Stimulation indices (SI)

Parameter

Negative control

50% test item

75% test item

100% test item

Positive control

Lymph node cell count

1.000

1.073

1.062

1.091

1.526**

Lymph node weight

1.000

1.137

1.235

1.176

1.471**

Ear weight

1.000

0.980

1.005

1.061

1.097**

Ear thickness (day 4)

1.000

0.996

1.008

0.988

1.118

**: significantly increased compared to negative control at p ≤0.01

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified