1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 May 2019 to 02 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 22 July 2010

Deviations:

yes

Remarks:

Minor deviations in animal housing temperature and relative humidity (due to summer heat wave and defective air humidifier) that did not affect the validity of the study (see section on environmental conditions)

GLP compliance:

yes (incl. QA statement)

Remarks:

Due to the short-term nature of the study no analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This exception is considered not to affect the purpose or integrity of the study.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

EC Number:

271-080-4

EC Name:

1,2-Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-products from

Cas Number:

68515-39-9

Molecular formula:

none available

IUPAC Name:

1,​2-​Benzenedicarboxylic acid, tridecyl ester, manuf. of, by-​products from

Specific details on test material used for the study:

Study substance: C13 Discard Alcohol (MRD-18-893)
Source: ExxonMobil Biomedical Sciences, Inc.
Batch number: 20181013
Expiration date of the batch: 13 Oct 2021
Purity: 100% (UVCB)
Physical state/Appearance: Colourless clear liquid
Storage conditions: At room temperature
Solubility and stability of the study substance in the vehicle: A solubility experiment was performed according to recommendations given by OECD 429; Study substance was soluble in the vehicle at concentrations up to 100%
Treatment of study substance prior to testing: used as received

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
Source: Envigo RMS B.V., Inc / Postbus 6174/ 5960 AD Horst / The Netherlands
Females nulliparous and non-pregnant: yes
Age at study initiation: 8 - 12 weeks
Weight at study initiation: 18.8 ± 1.3 g (Range: 15.8 – 20.9 g)
Housing: 4/cage
Diet: certified rodent diet, ad libitum
Water: tap water, ad libitum
Acclimation period: at least 5 days prior to dosing
Indication of any skin lesions: Only animals without any visible signs of illness were used for the study

ENVIRONMENTAL CONDITIONS
Temperature: 22 ± 2°C (except for deviations); actual 20-25°C
Humidity: relative humidity 45-65% (except for deviations); actual 19-70%
Photoperiod: 12 hrs light / 12 hrs dark

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

2.5%, 5%, 10%
The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS
Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest study substance concentration, which could be technically used was 100% (undiluted).
Irritation: Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% once daily each on three consecutive days. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin.
Systemic toxicity: At the tested concentrations the animals did not show any signs of systemic toxicity.
Ear thickness measurements: Ear thickness was determined prior to the first application of the study substance (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer. In addition, after the lymph nodes had been excised, both ears of mice were punched for determination of ear weights.
Erythema scores: The animals showed an erythema of the ear skin (up to Score 2), an erythema of the scalp, and scaly ears. Additionally, eschar formation of the ears and a gain of ear weight above the threshold of 25% was observed in both animals. In animal 2, additionally, a gain of ear thickness above the threshold of 25% was observed on day 6. Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. The animals showed an erythema of the ear skin (up to Score 1), an erythema of the scalp, and scaly ears. Additionally, the animal treated with 25% test item concentration showed eschar formation of the ears on the day of preparation, excluding this dose level for further use in the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Name of test method: Local Lymph Node Assay (LLNA); lymphocyte proliferation quantified by measuring the incorporation of radiolabelled thymidine into lymph node cells by β-Scintillation Counting
Criteria used to consider a positive response: A study substance is regarded as a sensitiser in the LLNA if the exposure to one or more test concentrations resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.).

TREATMENT PREPARATION AND ADMINISTRATION
Each experimental group of mice (n=4) was treated by topical application to the dorsal surface of each ear with study substance concentrations of 2.5, 5, or 10% in acetone/olive oil (4+1, v/v). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface of each ear once daily for three consecutive days. A control group of mice was treated with an equivalent volume of the vehicle alone. Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 19.8 µCi of 3H-methyl thymidine (equivalent to 79 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death. The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter.

OBSERVATIONS
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

Positive control substance(s):

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

Results and discussion

Positive control results:

Results from an experiment performed in April 2019 (Envigo study number 1950400) with positive control substance α-Hexylcinnamaldehyde at concentrations of 5, 10, and 25% in acetone:olive oil (4:1 v/v) produced Stimulation Index (S.I.) values of 1.48, 2.26, and 8.10, respectively. The calculated EC3 value was 11.3% (w/v).

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.44

Test group / Remarks:

2.5% test concentration

Remarks on result:

other: No indication of skin sensitisation

Results 2

Parameter:

SI

Value:

1.63

Test group / Remarks:

5% test concentration

Remarks on result:

other: No indication of skin sensitisation

Results 3

Key result

Parameter:

SI

Value:

1.23

Test group / Remarks:

10% test concentration

Remarks on result:

other: No indication of skin sensitisation

Cellular proliferation data / Observations:

DPM per lymph node:
0% test concentration (Control): 747.4
2.5% test concentration: 1074.9
5% test concentration: 1218.4
10% test concentration: 915.9

Any other information on results incl. tables

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The study substance was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of C13 Discard Alcohol, three groups each of four female mice were treated once daily with the study substance at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of four mice was treated with the vehicle (acetone/olive oil (4+1, v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. The animals treated with the study substance showed a very slight erythema of the ear skin (Score 1). 

A study substance is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of study substance required to produce a S.I. of 3 is referred to as the EC3 value.

In this study, Stimulation Indices of 1.44, 1.63, and 1.23 were determined with the study substance at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v). The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

The study substance was not a skin sensitiser under the test conditions of this study.