Butyl toluene-4-sulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2019 to 05 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Butyl toluene-4-sulphonate

EC Number:

212-295-5

EC Name:

Butyl toluene-4-sulphonate

Cas Number:

778-28-9

Molecular formula:

C11H16O3S

IUPAC Name:

butyl 4-methylbenzene-1-sulfonate

Test material form:

liquid

Remarks:

(colourless to slightly yellow)

Details on test material:

- Storage conditions: At room temperature protected from light

Specific details on test material used for the study:

- Purity/Composition correction factor: No correction factor required

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: inbred, SPF-quality
- Age at study initiation: Young adult animals (approximately 10 - 11 weeks old)
- Weight at study initiation: 18.7 - 24.6 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilised sawdust as bedding material and equipped with water bottles. For psychological/environmental enrichment, animals were provided with paper and shelters.
- Diet: pelleted rodent diet, ad libitum
- Water: municipal tap-water, ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 42 - 51 % (relative)
- Air changes: 10 or more air changes per hour with 100 % fresh air (no air recirculation)
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
- From: Not reported
- To: 03 February 2020

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

0 (vehicle control), 25, 50 and 100 % (w/w)

No. of animals per dose:

Five females per group

Details on study design:

PRE-SCREEN TESTS
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. Two test material concentrations were tested: 50 and 100 % concentrations. The highest concentration was the maximum concentration as required in the test guidelines. As hunched posture was noted in the main study following the first pre-screen test, another pre-screen was performed at test material concentrations of 50 and 100 % to clarify the clinical signs as seen for the main study.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
- Induction: Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test material.
- Excision of the Nodes: Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanised by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity: Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements: Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- Observations and measurements
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
All the animals were examined for reaction to dosing (once daily on Days 1-6; on Days 1-3 between 3 and 4 hours after dosing). The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing. Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) and scored as follows:
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth): 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4

No necropsy was performed, since all animals survived until the end of the observation period.

ANIMAL ASSIGNMENT
- Animal assignment: Animals were assigned to the study at the discretion of the co-ordinating biotechnician, with all animals within ± 20 % of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.

TREATMENT PREPARATION AND ADMINISTRATION
Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test material.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test material, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

DATA EVALUATION
DPM values were recorded for each animal and for each dose group. A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitiser.

Positive control substance(s):

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

Statistics:

Not performed

Results and discussion

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the Test Facility. In this study, performed in December 2019, females of the CBA/J mouse strain were checked for sensitivity to Alpha-Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD 429, EU Method B.42 and EPA OPPTS 870.2600. Alpha-Hexylcinnamaldehyde, technical grade was tested at concentrations of 5, 10 and 25 % in acetone/olive oil (4:1 v/v; AcOO) and afforded the following results:

0 % HCA: mean DPM ± SEM = 433 ± 73; SI ± SEM = 1.0 ± 0.2
5 % HCA: mean DPM ± SEM = 726 ± 55; SI ± SEM = 1.3 ± 0.3
10 % HCA: mean DPM ± SEM = 1481 ± 324; SI ± SEM = 3.4 ± 0.7
25 % HCA: mean DPM ± SEM = 2385 ± 583; SI ± SEM = 5.5 ± 1.3

The SI values calculated for the HCA concentrations 5, 10 and 25 % were 1.3, 3.4 and 5.5, respectively. An EC3 value of 9.0 % was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

1

Variability:

± 0.3 (Standard Error of the Mean)

Test group / Remarks:

vehicle control (0 % test material in vehicle)

Results 2

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

1.7

Variability:

± 0.3 (Standard Error of the Mean)

Test group / Remarks:

25 % test material in vehicle

Results 3

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

2

Variability:

± 0.2 (Standard Error of the Mean)

Test group / Remarks:

50 % test material in vehicle

Results 4

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

3

Variability:

± 0.7 (Standard Error of the Mean)

Test group / Remarks:

100 % test material in vehicle

Results 5

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

2.6

Test group / Remarks:

25 % test material in vehicle

Results 6

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

3.2

Test group / Remarks:

50 % test material in vehicle

Results 7

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

4.1

Test group / Remarks:

100 % test material in vehicle

Cellular proliferation data / Observations:

PRE-SCREEN TEST
Initially, a pre-screen test was performed at 50 and 100 % test material concentrations. Because no signs of systemic toxicity were noted and only very slight irritation was observed, a 100 % concentration was selected as highest concentration for the main study. The second pre-screen study confirmed the results of the first pre-screen test.

MAIN STUDY
Initially, a main study was performed by dosing three groups of five animals with one test material concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle. Unexpectedly, clinical signs (hunched posture on Days 2 and 3 for the majority of test material dosed animals and for one test material dosed animal on Day 4) were present in the animals of the main study and because the test guideline states that “doses should be used that do not induce systemic toxicity”, the results obtained were declared invalid and not used for interpretation. The main study was repeated using the same concentrations because the final results of the pre-screen study did indicate that no clinical signs were to be expected.

MACROSCOPIC EXAMINATION OF THE LYMPH NODES AND SURROUNDING AREA
The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal dosed at 50 % test material concentration and in all animals dosed at 100 % test material concentration, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX
Mean DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1282, 1458 and 2210 DPM, respectively. The mean DPM/animal value for the vehicle control group was 738 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 1.7, 2.0 and 3.0, respectively.
As the mean values showed a large variation, the SI values were also calculated based on the median values. Median DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1277, 1584 and 2022 DPM, respectively. The median DPM/animal value for the vehicle control group was 495 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 2.6, 3.2 and 4.1, respectively.
The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values.

CLINICAL OBSERVATIONS
- Mortality/Moribundity Checks: No mortality occurred.
- Clinical Observations (Post-dose Observations): No clinical signs of systemic toxicity were observed in the animals.
- Irritation: Very slight irritation of the ears was shown by all animals dosed at 100 and 50 % test material concentrations between Days 1 and 4; this was considered not to have a toxicologically significant effect on the activity of the nodes.
- Body weight: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sensitiser (Category 1B) according to EU criteria

Conclusions:

Findings from the study indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. The test material should therefore be classified as a skin sensitiser (category 1B) according to EU criteria.

Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600, and under GLP conditions.

Test material concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 25, 50 or 100 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (acetone/olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal dosed at 50 % test material concentration and in all animals dosed at 100 % test material concentration, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1282, 1458 and 2210 DPM, respectively. The mean DPM/animal value for the vehicle control group was 738 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 1.7, 2.0 and 3.0, respectively.

Because the mean values showed a large variation, the SI values were also calculated based on the median values. Median DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1277, 1584 and 2022 DPM, respectively. The median DPM/animal value for the vehicle control group was 495 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 2.6, 3.2 and 4.1, respectively.

The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

The results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. Accordingly, the test material is classified as a skin sensitiser (category 1B) according to EU criteria.