Registration Dossier

Administrative data

Description of key information

Skin sensitisation: SI value < 1 for all tested concentrations (BASF SE, 2016)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-08 to 2015-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Sample 14/078 from Mollescal HW, charge 10924344R0
- Expiration date of the lot/batch: April 28, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Species:
mouse
Strain:
CBA/Ca
Remarks:
OlaHs
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: 9 - 11 weeks
- Weight at study initiation: 19.7 - 21.5 g
- Housing: groupwise (Makrolon Type II (pre-test) / III (main study), with wire mesh top)
- Diet: ad libitum, 2018C Teklad Global 18% protein rodent diet
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65 %
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: ethanol/water (3+7, v/v)
Concentration:
5, 10, 25 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25 % solution in ethanol/water (3+7, v/v). Vortexing and slight warming to <40 °C were used to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. vortexing, sonicating, warming to 37 °C).
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals.
Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25 % once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. 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. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm²) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25 % was recorded on day 3 or day 6 (for detailed results see Appendix 1).
At the tested concentrations the animals neither showed any signs of local skin irritation nor systemic toxicity. Slight substance residuals were observed on the ears of both animals.
Thus, the test item in the main study was assayed at 5, 10, and 25 %. 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.

MAIN STUDY
Topical application
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25 % in ethanol/water (3+7, v/v). 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 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.

Determination of incorporated 3HTdR
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 per animal (2 nodes per animal). 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. 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.

Determination of Lymph Node Weight and Cell Count
After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (CASY® DT, Schärfe System). The values obtained were taken down manually.

Determination of Ear Weights
After the lymph nodes had been excised, both ears (left and right) 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 (pooled per animal) using an analytical balance.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and ethanol/water (3+7, v/v) was added. The different test item concentrations were prepared individually. The preparations were made freshly and used within two hours before each dosing occasion. Concentrations were in terms of material as supplied.
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, for the ear weights, the lymph node weights and lymph node cell count, and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between the test item groups and negative control group. For all statistical calculations validated statistical program R Script DecisionTree_2.Rnw was used. Statistical significance was set at the five per cent level (p < 0.05).
The Dean-Dixon-Test and Grubb’s Test were used for identification of possible outliers (performed with validated program R Script Outlier.Rnw). An outlier (DPM value for animal 14) was detected in the Grubb’s, but not in the Dean-Dixon-Test and was therefore not excluded from any calculations.
However, both biological and statistical significance were considered together.
Positive control results:
The positive control substance induced a statistically significant stimulation compared to the control.
Key result
Parameter:
SI
Value:
0.9
Test group / Remarks:
5 %
Key result
Parameter:
SI
Value:
0.95
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
1.68
Test group / Remarks:
25 %
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Radioactive labeling is used to measure proliferations.

EC3 CALCULATION
The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

CLINICAL OBSERVATIONS:
No deaths occurred during the study period. No signs of systemic toxicity or local skin irritation were observed during the study period. The animals of the high dose group showed slight substance residuals within the first hour after application from day 1 until day 3.

LYMPH NODE WEIGHTS AND CELL COUNTS
The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant or biologically relevant increase in lymph node weights or –cell counts was not observed in any of the test item treated groups in comparison to the vehicle control group. For BALB/c mice, a cut-off value for the lymph node cell count index of 1.55 was reported for a positive response. The indices determined for the lymph node cell count did not reach or exceed this threshold.

EAR WEIGHTS
The measured ear weight of all animals treated was recorded on test day 6 (after necropsy). A biologically relevant or statistically significant increase in ear weights was not observed. Furthermore, the cut-off value (1.1) of the ear weight index for a positive response regarding ear skin irritation reported for BALB/c mice was not reached or exceeded in any of the treated groups.

BODY WEIGHTS
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age. Some animals in the vehicle group and in the group treated with 5 % of test item showed a very minor decrease in body weight over the course of the study, which was biologically not relevant.
Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In this study the test item Disodium (sulphonatothio)acetate; dried was assessed for its skin sensitising potential using the Local Lymph Node Assay (LLNA) in mice (BASF SE, 58V0318/14X507, 2016). Test item solutions at different concentrations were prepared in the vehicle ethanol/water (3+7, v/v).

The local lymph node assay is recommended by international test guidelines (e.g., OECD) as an animal test for predicting skin sensitisation in humans and provides a rational basis for risk assessment. The basic principle underlying the LLNA is that sensitisers induce a primary proliferation of lymphocytes in the lymph node draining the application site. The ratio of proliferation in test item treated groups compared to that in vehicle controls is termed the Stimulation Index (S.I.). Radioactive labeling is used to measure cell proliferations.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could be technically achieved whilst avoiding systemic toxicity and excessive local skin irritation (as determined by a pre-experiment).

The animals showed neither signs of systemic toxicity nor local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant or biologically relevant increase in ear weights was not observed in any treated group in comparison to the vehicle control group. Furthermore, for BALB/c mice, a cut-off value of 1.1 for the ear weight index was reported for a positive response regarding ear skin irritation. None of the indices determined for the test item treated groups reached or exceeded this threshold.

A test item is regarded as a sensitiser in the LLNA if exposure to one or more test item concentration results 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 test item concentration required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices (S.I.) of 0.90, 0.95, and 1.68 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in ethanol/water (3+7, v/v), respectively. Thus, a clear dose response was observed.

A statistically significant and biologically relevant increase in DPM value and also in lymph node weight and -cell count was not observed in any dose group in comparison to the vehicle control group. Furthermore, the cut-off value of 1.55 for a positive response regarding the lymph node cell count index reported for BALB/c mice was not reached or exceeded in any dose group.

The test item Disodium (sulphonatothio)acetate; dried was thus not a skin sensitiser under the test conditions of this study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled as skin sensitising under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776. Further, no skin irritating properties were observed.