2-(4-Bromphenyl)-5-butyl-1,3-dioxane, trans-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 19 - Sep 26, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 429.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

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:

2-(4-Bromphenyl)-5-butyl-1,3-dioxane, trans-

EC Number:

817-763-8

Cas Number:

1310048-86-2

Molecular formula:

C14H19BrO2

IUPAC Name:

2-(4-Bromphenyl)-5-butyl-1,3-dioxane, trans-

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories GmbH, 5800 AN Venray, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: (20 ± 2) g
- Housing: Full barrier in an air-conditioned room
- Diet (e.g. ad libitum): Altromin 1324 ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

15.5, 25, and 50 % (v/v)

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO.

Preparation of the Test Item
Based on the results observed in the prescreen test the following test item concentrations were selected for the main study:
12.5%, 25% and 50% (v/v) The preparations were made immediately prior to each dosing.

Prescreen Test
Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals.
The maximum technically applicable concentration of the test item in the vehicle was found to be 50% in AOO (Acetone, Sigma-Aldrich, lot no. STBJ1392, expiry date: 01/06/2020; olive oil highly refined).
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) day 6. Excessive local irritation was indicated by an erythema score ≥ 3 and/or ear swelling of ≥ 25%.

Controls
AOO was used as vehicle and served as negative control.
Positive controls are performed periodically. The recent results are included in the report.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: random
- Criteria used to consider a positive response: cf. OECD 429

Preparation of the Animals
The animals were randomly selected using the validated departmental computerised system E WorkBook (latest version, ID Business Solutions Ltd.).
Identification was ensured by cage number and individual marking (tail).

Clinical Observation
Prior to the application and once a day thereafter all animals were observed in order to detect signs of toxicity, including dermal irritation at site of application.

Weight Assessment
The animals were weighed prior to the application and at the end of the test period (prior to the treatment with 3HTdR).

Dose Groups
3 test groups (3 different concentrations) and 1 negative control group (vehicle) were tested

Topical Application
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear.
Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methyl thymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated.
After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4° C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H -Methyl Thymidine
The 3H-methyl thymidine – incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methyl thymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

Evaluation of Results
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.
EC3 values, calculated concentrations which induce stimulation indices of three, are determined by linear interpolation, EC3=c+[(3-d)/(b-d)]x(a c), between two points of the stimulation indices axis, one above (a,b) and one below (c,d) the stimulation index of three. If all measured points are above or below the stimulation index of three, no EC3 value can be stated.
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0).
On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011 as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017:

Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser
a) if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or
b) if there are positive results from an appropriate animal test.
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value ≤ 2%
WARNING, exclamation mark. May cause an allergic skin reaction.
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%
WARNING, exclamation mark. May cause an allergic skin reaction.

Positive control substance(s):

other: shared control 1% phenylenediamine

Statistics:

Standard statistical methods have been applied for data processing.

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.9

Variability:

0.4

Test group / Remarks:

12.5 %

Results 2

Key result

Parameter:

SI

Value:

1.9

Variability:

0.3

Test group / Remarks:

25 %

Results 3

Key result

Parameter:

SI

Value:

3.9

Variability:

0.8

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION

EC3 CALCULATION

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any signs of systemic toxicity or excessive local irritation. Minor local effects such as sticky or scrubby fur were observed in all animals treated with the test substance from day 2 (second dosing) until day 6 (last day of observation). Temporarily, all animals of the 25% test group showed desquamation on day 2.

BODY WEIGHTS
All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Measurement of Ear Thickness

Directly prior to the first application, approximately 48 hours after the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured.
The means of the ear thickness per group showed no relevant difference compared to the negative control.

Mean ear thickness on	Day 1	Day 3	Day 6
12.5% test group was	0.16 mm	0.17 mm	0.17 mm
25% test group was	0.17 mm	0.17 mm	0.17 mm
50% test group was	0.16 mm	0.17 mm	0.17 mm
negative control group	0.17 mm	0.17 mm	0.17 mm

Conclusion

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 38.81%.
The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group.
Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers.
According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 38.81%. The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group. Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.

Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 429. The test material was a skin sensitiser under the test conditions of this study. Based on the results of the prescreen test the test item was assessed for sensitising properties at concentrations of 12.5%, 25% and 50% (w/v), each dissolved in AOO 4:1 (v/v).

There was no mortality and there were no significant clinical observations or effects on body weights. The EC3 value (derived by linear interpolation) was calculated to be at a test item concentration of 38.81%. The measurement of the ear thickness did not reveal any relevant increase of the ear thickness in any dosage group. Consequently, according to OECD 429 solutions or preparations containing more than 38.81% test material are expected to have a stimulation index of >3 and are therefore considered to be dermal sensitisers. According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has obligatory labelling requirement for skin sensitisation and is classified into Category 1B.