Dodecane-1,12-diol

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

yes

Remarks:

- modified LLNA = Local Lymph Node Assay (LLNA) - Integrated Model for Differentiation of Skin reactions (IMDS)): Measurement of cell counts instead of radioactive labeling. In addition, measurements of ear swelling and ear weights were done to discrimina

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 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).
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 of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
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. 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.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dodecane-1,12-diol

EC Number:

227-133-9

EC Name:

Dodecane-1,12-diol

Cas Number:

5675-51-4

Molecular formula:

C12H26O2

IUPAC Name:

dodecane-1,12-diol

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Due to the limited solubility, the test item was dissolved in dimethyl sulfoxide (DMSO) and heated up to 37 °C to gain the maximal feasible concentration of 10% (w/w).
DMSO was selected as vehicle as it provided a suitable solution of the test item both for administration and adherence to the mouse ear and is recommended by the OECD guideline. Acetone/olive oil (4:1, v/v), methyl ethyl ketone, propylene glycol and N,N-dimethylformamide, other recommended vehicles, did not provide higher concentrated suitable solutions or suspensions.

- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
The analysis of the test item/vehicle solutions of the 2.5%, 5% and 10% (w/w) concentrations for the actual test item levels was carried out (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 97.4 – 98.6% (2.5% (w/w) concentration), 96.1 – 98.5% (5% (w/w) concentration) and 97.4 – 100.2% (10% (w/w) concentration) of the nominal concentration.

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, Sanhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 26-34-g
- Housing:
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C  3°C (maximum range)
- Humidity (%): 55%  10% (maximum range)
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 h/12 h
- IN-LIFE DATES: From: To: 07 November - 05 December 2019

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 2.5, 5, 10 %

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1:
The weight and any signs of toxicity of each animal were determined and 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):
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.
Observations:
- 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
- ear swelling
- ear weight
- body weights
- clinical signs

PRE-SCREEN TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations (w/w), 2.5%, 5% and 10% dissolved in DMSO 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 3 animals (one per concentration) were used.
- Irritation: No irritating properties were observed in this preliminary experiment at concentrations of 2.5%, 5% and 10%, no differences in ear weight and ear thickness were noted.
- Systemic toxicity: none
- Ear thickness measurements: yes

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For lymph node weight a significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY). A possible concentration-response-relationship was examined by linear regression analysis employing PEARSON's correlation coefficient. An U-test was performed also for the cell count.
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.

Results and discussion

Positive control results:

Alpha hexyl cinnamic aldehyde (20% solution) shows a clear sensitizing potential in the local lymph node assay (IMDS).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

Stimulation Index

Value:

1

Test group / Remarks:

vehicle control group

Remarks on result:

other: no skin sensitization

Results 2

Parameter:

SI

Remarks:

Stimulation index

Value:

0.996

Test group / Remarks:

2.5% test item

Remarks on result:

other: no skin sensitization

Results 3

Parameter:

SI

Remarks:

Stimulation index

Value:

1.078

Test group / Remarks:

5% test item

Remarks on result:

other: no skin sensitization

Results 4

Parameter:

SI

Remarks:

Stimulation index

Value:

0.985

Test group / Remarks:

10% test item

Remarks on result:

other: no skin sensitization

Any other information on results incl. tables

In the main study treatment with 1,12-Dodecanediol at the concentration of 2.5%, 5% and 10% test item did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation indices of the lymph node cell count did not exceed the threshold level of 1.4.

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 at any concentration.

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.

No signs of local or systemic intolerance were recorded.

parameter	control group (vehicle)	2.5% test item	5% test item	10% test item	positive control goup
lymph node cell count	1.000	0.996	1.078	0.985	1.602**
lymph node weight	1.000	1.036	1.164	1.291	1.519**
ear weight	1.000	0.939	0.862	0.981	1.065
ear thickness TD4	1.000	0.988	0.984	0.981	1.093

Applicant's summary and conclusion

Interpretation of results:

other: no sensitizing potential; no indication for non-specific (irritant) activation

Executive summary:

The registered substance showed no sensitizing potential in the modified Local Lymph Node Assay (IMDS, following OECD TG 429) in female NMRI mice after dermal application of up to and including the maximum technically feasible concentration of 10 %. Additionally, no indication for a non-specific (irritant) activation was detected.

In conclusion, under the present test conditions, 1,12-Dodecanediol at the concentration of 2.5%, 5% or 10% (w/w) did not reveal any skin sensitising properties in the local lymph node assay and therefore 1,12-Dodecanediol is not classified to be skin sensitising in this test system.