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Administrative data

Description of key information

Skin sensitisation (OECD 429), LLNA: not sensitising

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:
01 Jun - 27 Oct 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
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 Verbrauche rschutz, Hamburg, Germany
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, 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: 27 - 36 g (test day 1)
- Housing: animals were kept individually in in MAKROLON cages (type II) 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
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50, 75 and 100% (experiment I)
25% (experiment II)
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 (experiment I). The concentrations (50, 75 and 100%) were chosen based on a preliminary experiment. In addition, three further groups of 6 female animals treated with a 25% concentration of the test item, vehicle or positive control (experiment II) were examined based on strong irritating properties of the test item at concentrations of 50, 75 and 100% (experiment I). 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.
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.
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.183
Test group / Remarks:
50% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.513
Test group / Remarks:
75% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.698
Test group / Remarks:
100% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.701
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment I
Key result
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.033
Test group / Remarks:
25% test item
Remarks on result:
other: no indication of skin sensitisation
Remarks:
Experiment II
Parameter:
SI
Remarks:
Lymph node cell count
Value:
1.839
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment II
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.023
Test group / Remarks:
50% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.442
Test group / Remarks:
75% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.674
Test group / Remarks:
100% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.721
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment I
Key result
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.038
Test group / Remarks:
25% test item
Remarks on result:
other: no indication of skin sensitisation
Remarks:
Experiment II
Parameter:
SI
Remarks:
Lymph node weight
Value:
1.827
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment II
Parameter:
SI
Remarks:
Ear weight
Value:
1.167
Test group / Remarks:
50% test item
Remarks on result:
other: indication of skin irritation
Remarks:
Experiment I
Parameter:
SI
Remarks:
Ear weight
Value:
1.132
Test group / Remarks:
75% test item
Remarks on result:
other: indication of skin irritation
Remarks:
Experiment I
Parameter:
SI
Remarks:
Ear weight
Value:
1.282
Test group / Remarks:
100% test item
Remarks on result:
other: indication of skin irritation
Remarks:
Experiment I
Parameter:
SI
Remarks:
Ear weight
Value:
1.241
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment I
Key result
Parameter:
SI
Remarks:
Ear weight
Value:
1.012
Test group / Remarks:
25% test item
Remarks on result:
other: no indication of skin irritation
Remarks:
Experiment II
Parameter:
SI
Remarks:
Ear weight
Value:
1.244
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment II
Parameter:
SI
Remarks:
Ear thickness
Value:
1.052
Test group / Remarks:
50% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Ear thickness
Value:
1.087
Test group / Remarks:
75% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Ear thickness
Value:
1.113
Test group / Remarks:
100% test item
Remarks on result:
other: Experiment I
Parameter:
SI
Remarks:
Ear thickness
Value:
1.165
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment I
Key result
Parameter:
SI
Remarks:
Ear thickness
Value:
1.008
Test group / Remarks:
25% test item
Remarks on result:
other: no indication of skin irritation
Remarks:
Experiment II
Parameter:
SI
Remarks:
Ear thickness
Value:
1.198
Test group / Remarks:
Positive control
Remarks on result:
other: Experiment II
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 undiluted test item revealed statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count exceeded the threshold level of 1.4 at a concentration of 75% in acetone/olive oil (4:1, v/v) (w/w) or the undiluted test item. In addition, the lymph node weight was significantly increased for the concentrations of 75% and 100% of the test item. As the stimulation indices of ear weight exceeded the threshold level of 1.1 at concentrations of 50% and above, the test item was considered to have strong irritating properties in this concentration range in this test system. At the concentration of 25% neither sensitising nor irritating properties were observed.

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

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

Table 1: Summary of Stimulation indices (SI)

 

Experiment I

Experiment II

Parameter

Negative

control

50% test item

75% test item

100% test item

Positive control

25% test item

Negative control

Positive control

Lymph node cell count

1.000

1.183

1.513

1.698**

1.701**

1.033

1.000

1.839**

Lymph node weight

1.000

1.023

1.442**

1.674**

1.721**

1.038

1.000

1.827**

Ear weight

1.000

1.167**

1.132**

1.282**

1.241**

1.012

1.000

1.244**

Ear thickness (day 4)

1.000

1.052

1.087

1.113

1.165

1.008

1.000

1.198

** significantly increased compared to control at p ≤0.01

Interpretation of results:
GHS criteria not met
Conclusions:
CLP: not classified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation

The skin sensitising properties of Fatty acids, C8-10 (even numbered), diesters with neopentyl glycol and di- and triesters with trimethylolpropane (CAS 97281-24-8) were tested in a study according to OECD guideline 429 and in compliance with GLP (Oleon, 2016) using the modified local lymph node assay (LLNA). In this study groups of six female NMRI mice were treated with the test item at concentrations of 50, 75 and 100% (w/w) in acetone/olive oil (4:1 v/v) (experiment I). Topical application of 25 µL of the appropriate test substance concentrations was performed daily at the dorsal surface of each ear for three consecutive days. Four days following the first topical application of the test item or vehicle ear swelling measurements of all mice (immediately before sacrificing the mice) were carried out at the helical edge of both ears followed by lymph node cell count, lymph node weight and ear weight measurements after animal sacrifice. Positive and negative controls were included in the study and gave the expected results. Treatment with the undiluted test item revealed statistical significantly increased values for the lymph node cell count. The stimulation indices of the lymph node cell count exceeded the threshold level of 1.4 at a concentration of 75% (w/w) in acetone/olive oil (4:1, v/v) or the undiluted test item. In addition, the lymph node weight was significantly increased for the concentrations of 75% and 100% (w/w) of the test item. As the stimulation indices of ear weight exceeded the threshold level of 1.1 at concentrations of 50% (w/w) and above, the test item was considered to have strong irritating properties in this concentration range in this test system. Therefore, an additional group of six female NMRI mice was treated with the test item at a concentration of 25% (w/w) in acetone/olive oil (4:1 v/v) in a second experiment following the same experimental protocol as described above. At the concentration of 25% (w/w) neither sensitising nor irritating properties were observed. The calculated stimulation indices (SI) of the test substance are 1.033 (lymph node cell count), 1.038 (lymph node weight), 1.012 (ear weight) and 1.008 (ear thickness) at a concentration of 25% (w/w) and therefore <1.4 (lymph node cell count and lymph node weight) and <1.1 (ear weight), respectively. Thus, under the conditions of the test, the test substance revealed no skin sensitising and irritating properties at a concentration of 25% (w/w).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.