3-tert-butyladipic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-11-28 to 2016-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 22, 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 20, 2012

Deviations:

no

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:

3-tert-butyladipic acid

EC Number:

233-759-3

EC Name:

3-tert-butyladipic acid

Cas Number:

10347-88-3

Molecular formula:

C10H18O4

IUPAC Name:

3-tert-butylhexanedioic acid

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Changzhou Sunlight Pharmaceutical Co., Ltd., China; 20160505 / 116727
- Expiration date of the batch: May 2017
- Purity test date: 99.72 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

Ola Hsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: good conventional
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: 16.8 - 21.3 g
- Housing: Grouped caging (4 animals/cage)
- Diet (e.g. ad libitum): ad libitum, ssniff® Rat/Souris-Elevage E complete diet (ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 – 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

5, 10, 25, 50 % (w/v)

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Yes
- Irritation: Yes
- Systemic toxicity: Yes
- Ear thickness measurements: Yes
- Erythema scores: Yes

Solubility of the test item was evaluated in the following vehicles (listed in order of preference) at concentration of 10 % (w/v) and above using concentration series of 100 %, 50 %, 25 %, 10 %, 5 % etc. according to the relevant guidelines.
1. Acetone: Olive oil 4:1 (v/v) mixture (AOO)
2. N,N-Dimethylformamide (DMF)
3. Dimethyl sulfoxide (DMSO)
4. Methyl ethyl ketone (MEK)
5. Propylene glycol (PG)
6. Aqueous 1 % (w/v) Pluronic®PE 9200 (Plu)
7. Absolute ethanol: water 7:3 (v/v) mixture (EtOH)

The best solubility was achieved in DMF and DMSO with a maximum concentration of 50 % (w/v). Less or no solubility was observed with the other vehicles. As DMF is more preferred than DMSO the test item was formulated with the following vehicle in the main test.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity were observed. No sign of significant irritation (indicated by an erythema score ≥ 3 and/or an increase of ≥ 25 % of ear thickness observed on any day of measurement) or any other local effect was observed.

MAIN STUDY
In vivo treatment
Animals in the treatment groups were treated with the relevant vehicles (DMF or AOO), appropriate formulations of the test item or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to the results of the DRF test.
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There were no treatments on Days 4, 5 and 6.

Proliferation assay
No technical failure was observed during the test. No animals showed symptoms of systemic toxicity or sign of excessive skin irritation hence all treated animals were processed.
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 μCi# of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).

Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.

Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation, the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.

Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants. Then the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the β-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA.

Clinical Observations
During the main test (from Day 1 to Day 6) all animals were observed at least once a day for any clinical signs, including systemic toxicity and local irritation. Irritation was monitored by erythema scoring during the whole test. Individual records were maintained.

Body Weight
Individual body weights were recorded on Day 1 (beginning of the test) and on Day 6 (prior to 3HTdR injection) with a precision of ± 0.1 g.

Evaluation of the Results
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value: the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed as DPM/group and as DPM/mouse.
The stimulation index (SI = the DPM/mouse of a treated [positive control or test item] group divided by the DPM/mouse of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Significance of the dose-response was evaluated by linear regression using the calculated SI values. All calculations were made by Microsoft Excel Software. Based on the results, the EC3 value (dose calculated to induce a stimulation index of 3) of the test item was not calculated.

Criteria used to consider a positive response
The test item is considered as a skin sensitizer, if the following criterion is fulfilled:
- Exposure to at least one concentration (non-irritating, non-toxic) of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control group animals were treated with 25 % HCA solution (formulated in AOO) concurrent to the test item treated groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. The positive control substance induced the appropriate stimulation compared to the relevant control (AOO). The calculated SI value was 7.3. The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

7.3

Test group / Remarks:

Positive control 25 % HCA in AOO

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

Vehicle control for the test item

Results 3

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

50 % in DMF

Results 4

Parameter:

SI

Value:

1.6

Test group / Remarks:

25 % in DMF

Results 5

Parameter:

SI

Value:

1.4

Test group / Remarks:

10 % in DMF

Results 6

Parameter:

SI

Value:

2.8

Test group / Remarks:

5 % in DMF

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No treatment group was excluded from the evaluation since no failed treatment, sign of systemic toxicity or irritation was observed during the test.
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control only. Appearance of the lymph nodes was normal in the negative control groups (AOO or DMF) and in the test item treated groups.
No significant lymphoproliferative response (SI ≥ 3) was observed for the test item at the tested concentrations. The observed stimulation index values were 1.6, 1.6, 1.4 and 2.8 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively.
Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.52, r = 0.48).

CLINICAL OBSERVATIONS
No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score ≥ 3) or any other local effect were observed in any treatment group.

BODY WEIGHTS
No significant, treatment related effect on the body weights was observed in any treatment group. Loss of body weight ≥ 5 % was observed in the positive control group and in the 10 % (w/v) dose group (1 of the 4 animals in both groups; 6 % or 5 % decrease, respectively) but the mean body weights did not decrease hence the effect was considered not significant.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance does not show any skin sensitising properties.

Executive summary:

The aim of this study was to determine the skin sensitization potential of 3-Tert Butyladipic Acid following dermal exposure in the Local Lymph Node Assay according to OECD guideline 429. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and a Dose Range Finding test (DRF). Solubility of the test item in vehicles preferred in the LLNA was evaluated. Based on the results the test item was formulated in N,N-Dimethylformamide (DMF) in the LLNA. The maximum attainable concentration (based on solubility) was 50 % (w/v). According to results of the DRF, where no adverse effect was observed up to this maximum soluble concentration, the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % or 5 % (w/v).

Appropriate positive control (α-Hexylcinnamaldehyde, HCA), furthermore two negative control groups dosed with the vehicles of the test and positive control groups, respectively, were employed.

The positive control item [25 % (w/v) HCA in Acetone:Olive oil 4:1 (v/v) mixture, AOO] induced significant stimulation over the relevant control (SI = 7.3) thus confirming the validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

No significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the relevant control (DMF) was noted for 3-Tert Butyladipic Acid at the applied test concentrations. The observed stimulation index values were 1.6, 1.6, 1.4 and 2.8 at test item concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. No significant dose-response relationship was observed (p = 0.52, r = 0.48, evaluated by linear regression using the SI values). Although the SI value of 2.8 observed at the lowest tested concentration (5 %, w/v) was close to the threshold value of 3 it was considered to be not biologically relevant since no significantly increased, dose-related lymphoproliferation was observed at the higher test concentrations.

According to evaluation criteria of the relevant guidelines, the lack of a significantly increased lymphoproliferation (indicated by an SI ≥ 3) up to the maximum attainable concentration of 50 % (w/v, based on solubility) and also the lack of a significant dose-response relationship are considered as evidence that 3-Tert Butyladipic Acid is not a skin sensitizer.

Under the conditions of the present assay, 3-Tert Butyladipic Acid tested at the maximum feasible concentration of 50 % (w/v, based on solubility) and also at concentrations of 25 %, 10 % or 5 % (w/v) as formulations (apparently solutions) in a suitable vehicle (N,N-Dimethylformamide) was shown to have no skin sensitization potential in the Local Lymph Node Assay.