Sulfonic acids, shale-oil, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

initiated: 2016-08-22, experimental: 2017-03-08 to 2017-04-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

OECD 429 testing has been considered to be best for this type of substance. In addition the study has been commissioned before Regulation (EU) 2016/1688 entered into force. The delay of the experimental study was due to a change of testing facility.
Sensibilisation studies according to OECD 442C (KeratinoSens) and OECD442D (h-CLAT) have been considered but finally rejected, since they appear not suitabel for week sensitizers and may lead to false negative results.

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:

2010

Deviations:

yes

Remarks:

The age of the animals was 12 to 13 weeks (beginning of acclimatisation). This deviation to the study plan, however, does not affect the validity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

The age of the animals was 12 to 13 weeks (beginning of acclimatisation). This deviation to the study plan, however, does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, date of inspection: 13.-16. Juli 2015

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Sulfonic acids, shale-oil, sodium salts

EC Number:

297-668-0

EC Name:

Sulfonic acids, shale-oil, sodium salts

Cas Number:

93686-18-1

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

Sulfonic acids, shale-oil, sodium salts

Test material form:

liquid - solid: mixture of

Remarks:

aqueous solution

Details on test material:

Tiroler Steinöl sulfoniert D90 (TS-D90)

Specific details on test material used for the study:

technical grade: aqueous solution (31% dry matter in water)

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Remarks:

Recognised as the recommended test system.

Sex:

female

Details on test animals and environmental conditions:

Source: Envigo RMS B.V., Inc, Postbus 6174, 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (nulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Pre-test: 12 - 13 weeks
Main study: 9 - 10 weeks
Body weight: 19.3 ± 0.3 g (start),
19.7 ± 1.1 g (prior administration);
see Appendix 1 and 2 of the study report
Identification:
The animals were distributed into the test groups at random. All animals belonging to the same experimental group were kept in one cage. In the main experiment, the animals were identified by fur clipping. In the pre-experiment, animals were identified by cage number.

Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity: approx. 45-65%
artificial light: 6.00 a.m. - 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

other: Ethanol/water (3+7, v/v)

Remarks:

Ethanol: Purity: ≥99.9%; Water: Sterile

Concentration:

0, 25, 50, and 100%

No. of animals per dose:

4

Details on study design:

TEST ITEM ADMINISTRATION
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% in ethanol/water (3+7, v/v), and 100%. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF ³H-METHYL-THYMIDINE
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 20.1 μCi of 3H-methyl thymidine (equivalent to 80.4 μCi/mL 3HTdR) were
injected into each test and control mouse via the tail vein.

TERMINAL PROCEDURE
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).

PREPARATION OF SINGLE CELL SUSPENSIONS
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.

DETERMINATION OF CELLULAR PROLIFERATION (incorporation of ³HTdR)
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. Similarly, background ³HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses ³HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS
Clinical 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.
Determination of Ear Thickness: In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of ear weights: In the pre-test, after the lymph nodes had been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
Determination of Body Weights: The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment)

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables. Where appropriate, the EC3 value were calculated according to the equation

EC3 = (a-c) [(3-d)/(b-d)] + c

where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the coordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot. All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Results and discussion

Positive control results:

RESULTS OF THE GLP POSITIVE CONTROL
Experiment performed in October 2016 (Envigo study number 1792800). Positive control substance: α-Hexylcinnamaldehyde Vehicle: acetone:olive oil (4+1, v/v)

Test Item [%] Stimulation Index [S.I.]
0 1.00
5 1.78
10 3.43
25 10.06

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0

Test group / Remarks:

1

Results 2

Key result

Parameter:

SI

Value:

1.74

Test group / Remarks:

2

Remarks on result:

other: 25% in ethanol/water (3+7, v/v)

Results 3

Key result

Parameter:

SI

Value:

1.57

Test group / Remarks:

3

Remarks on result:

other: 50% in ethanol/water (3+7, v/v)

Results 4

Key result

Parameter:

SI

Value:

4.18

Test group / Remarks:

4

Remarks on result:

other: 100% in ethanol/water (3+7, v/v)

Results 5

Parameter:

other: DPM per lymph node

Value:

551.2

Test group / Remarks:

1

Results 6

Parameter:

other: DPM per lymph node

Value:

960.8

Test group / Remarks:

2

Remarks on result:

other: 25% in ethanol/water (3+7, v/v)

Results 7

Parameter:

other: DPM per lymph node

Value:

866.6

Test group / Remarks:

3

Remarks on result:

other: 50% in ethanol/water (3+7, v/v)

Results 8

Parameter:

other: DPM per lymph node

Value:

2 303.7

Test group / Remarks:

4

Remarks on result:

other: 100% in ethanol/water (3+7, v/v)

Cellular proliferation data / Observations:

It cannot be ruled out that the physical damage on the ear skin observed in high dose animals had a possible effect on S.I. value in this dose group. The lesions could have caused mild unspecific proliferation of lymphocytes, leading to a weakly positive S.I. value in the high dose only. Furthermore, no conventional dose response was obtained.

Any other information on results incl. tables

EC3 Value

An EC3 value could not be derived since no conventional dose response was obtained and the

results were found to be inconclusive due to substantial skin irritation in high dose animals.

Deaths

No deaths occurred during the study period.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The results obtained with sulfonic acids, shale oil, sodium salts, were found to be inconclusive under the test conditions of this study since a possible effect of skin irritation on lymphocyte proliferation in high dose animals cannot be ruled out.

Executive summary:

In the study the test item sulfonic acids, shale oil, sodium salts, formulated in ethanol/water (3 +7, v/v) was assessed for its possible skin sensitising potential. For this purpose a local lymph node assay was performed using test item concentrations of 25, 50% (w/w), and 100%.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with a test item concentration of 50 and 100% showed a very slight erythema of the ear skin (Score 1) on few days. Additionally hair loss was observed (see Appendix 2 for details). Animals treated with 25% test item concentration did not show any signs of local skin irritation. On test day 6, upon preparation, focal eschar (crust) formation was observed on the ears of the animals treated with the undiluted test item. This minor physical damage of the skin was possibly caused by attempted removal of adhered test item.

In this study Stimulation Indices (S.I.) of 1.74, 1.57, and 4.18 were determined with the test item at concentrations of 25 and 50% in ethanol/water (3+7, v/v), as well as undiluted (100%), respectively. It cannot be ruled out that the physical damage on the ear skin observed in high dose animals

had a possible effect on S.I. value in this dose group. The lesions could have caused mild unspecific proliferation of lymphocytes, leading to a weakly positive S.I. value in the high dose only. Furthermore, no conventional dose response was obtained. The results obtained with sulfonic acids, shale oil, sodium salts, were thus found to be inconclusive.