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Description of key information

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:
24-29 Oct 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Remarks:
TNO Triskelion, Utrechtseweg 48 3704 HE Zeist, the Netherlands
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approximately 9-11 weeks
- Weight at study initiation: 21.1 - 25.6 g
- Housing: During acclimatisation the animals were group housed in macrolon type III cages (maximally 5 mice/cage). After allocation the animals were housed in macrolon type III cages (4 mice/group/cage) for the main study. For the dose range finding test, one animal was housed in a macrolon type II cage. The cages were provided with wood shavings (Lignocel) as bedding material and shreds of paper (Enviro-dri) and gnaw wood as environmental enrichment
- Diet: Standard diet ad libitum (Rat and Mouse no.3 breeding diet RM3) was purchased from SDS Special Diets Services, Whitham, England.
- Water: domestic mains tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2, one deviation from the optimal temperature was observed (period of <30 minutes of 24.7°C).
- Humidity (%): 45-65, the actual upper limit during the study period was 99.9%, which was considered not to have affected study integrity.
- Air changes (per hr): ca 10
- Photoperiod (hrs dark / hrs light): 12/12 (light on from 7:00 AM - 7:00 PM)
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 5, 10, 25% w/v
No. of animals per dose:
4
Details on study design:
DOSE FORMULATIONS:
The dose formulations of the test substance in vehicle were freshly prepared on each day of dosing, i.e. day 0, 1 and 2. The formulation was prepared on a weight (test substance)/volume (vehicle) basis. The formulation was mixed (vortex) until visible homogeneity was obtained. Dose formulations were prepared at concentrations selected from the series: 50% and 25% (dose range finding study) and 25%, 10% and 5% (main study). No adjustment was made for the purity of the test substance. The dose formulations were used for dosing of the animals within 2 hours after preparation. Homogeneity, concentration and stability of the test substance in vehicle were not determined in this study.

STUDY DESIGN:
- Dose range finding test: One randomly selected animal was treated in the dose range finding test, each ear with a test substance concentration from the series: 50 and 25%. The test substance formulations were applied on the dorsum of the ears (25 μL on each ear) for three consecutive days, i.e. days 0, 1 and 2. The ears of the animal were observed daily for signs of irritation. Moreover, the animal was also checked for clinical signs and changes in body weight. The animal was sacrificed approximately 24 hours after its third treatment on day 3.
- Main study: On day 0, 1 and 2 the different concentrations of test substance and control substances were administered to the dorsum of both ears (25 μL on each ear by means of a pipette). On day 5 all animals received an intravenous injection of 250 μL of phosphate-buffered saline (PBS) containing 20 μCi of 3H-thymidine in the tail vein. Five hours after the 3H-thymidine injection the animals were sacrificed by intra peritoneal (i.p.) injection with an overdose of sodium pentobarbital and the draining auricular lymph nodes were excised, pooled per animal in PBS and further processed to determine the 3H-thymidine incorporation. Per animal, the paired auricular lymph nodes (ALN) were excised and were placed together in a vial containing 5 mL phosphate buffered solution (PBS). A single cell suspension was prepared by gently rubbing the nodes between the rough ends of microscope slides. The cell suspension was washed twice. The cell pellet obtained after the second wash was resuspended in 3 mL of 5% trichloroacetic acid (TCA) and left overnight (~18 hours) at 2-10ºC. After centrifugation and removal of the supernatant, 1 mL of 1.5 M KOH in 20% EtOH was added to the precipitate and left for 24 hours at room temperature. Thereafter, the solution was transferred into a clean scintillation vial and 20 mL liquid scintillation cocktail (Hionic Fluor, Perkin Elmer) was added. The vial was thoroughly shaken and the number of disintegrations per minute (DPM) was determined by counting for five minutes in a liquid scintillation counter (Perkin Elmer).

OBSERVATIONS:
The general condition and behaviour of the animals were checked at least once daily during the study. All findings including any abnormalities were recorded in the morning, when applicable before dosing and in case of changes in clinical signs also in the afternoon. Body weights were determined at allocation, at the start (day 0) and at the end (day 5) of the study. The animals were sacrificed on day 5 for excision of the auricular lymph nodes. The animals were macroscopically examined including the auricular lymph nodes and the ears.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical evaluation of the data (body weights and 3H-thymidine incorporation) were performed by one-way ANOVA, followed by Dunnett’s multiple comparison tests in case of a significant result. In case of difference of variance between the different groups, log-transformation of the data before statistical evaluation was considered. Body weights and 3H-thymidine incorporation of the test groups 2, 3 and 4 or the positive control group were compared with the vehicle control animals. Probability values of p<0.05 were considered significant.
Positive control results:
The stimulation index of Hexyl Cinnamic Aldehyde (25% in vehicle v/v) was 4.42.
Parameter:
SI
Remarks:
Concentration 0 % w/v
Value:
1
Parameter:
SI
Remarks:
Concentration 5 % w/v
Value:
11.82
Parameter:
SI
Remarks:
Concentration 10 % w/v
Value:
23.13
Parameter:
SI
Remarks:
Concentration 25 % w/v
Value:
17.38
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/v) Disintegrations per minute (group means) 0 : 1672 5 : 19763 10 : 38675 25 : 29067

- Dose range finding test: Application of a 50% Tosyl chloride formulation resulted in redness of the skin of the (left) ear. The redness had not resolved before application of the next dose (approximately 24 hours after the previous) and persisted for at least 24 hours after the last (third) dosing. No indication of an increase in the degree of irritation was observed after repeated application. The right ear, dosed with a 25% Tosyl chloride formulation, showed similar redness and was also persistent for at least 24 hours after the last (third) application. Comparable with the 50% concentration, no indication of an increase in the degree of irritation was observed after repeated application of the 25% concentration. After the third dosing, redness was observed in the skin on the head between both ears, which had disappeared within 24 hours. This was considered a spread of the signs of irritation outside the area of application. The signs of irritation were indicative of absorption of the test substance through the skin, and the results showed that there was no (clear) difference in degree of irritation between a 50% and 25% formulation. To prevent an interference of the irritable state of the animal on the stimulation of the proliferation of lymphocytes in the auricular lymph node as much as possible, a 25% concentration was selected as the highest concentration to be tested in the main study. The concentrations to be used in the main study were thus selected to be 25%, 10% and 5% Tosyl chloride (w/v).

- Main study: Signs of erythema of the ears were observed in all animals of groups 3 and 4. In the animals of group 3, erythema of the ears was observed from day 2 until the end of the study period (day 5), whereas in group 4 erythema was observed from day 0 until the end of the study period. No aberrant body weights or body weight gains were observed and no statistical significant difference was found between the vehicle and the different test groups and between the vehicle and the positive control group.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions of this study, evidence was obtained that Tosyl chloride has sensitising potential when dermally applied at concentrations of 5% and higher.
Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of Tosyl chloride were studied in mice. Three test groups of 4 female mice each were treated with 5, 10, and 25% w/v by means of open application of 25 μL to the dorsum of each ear (total 50µL) for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H-thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a negative control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v). Group mean 3H-thymidine incorporations of 19763, 38675 and 29067 DPM were found in the auricular lymph nodes of animals treated with respectively 5, 10 and 25% Tosyl chloride. The group mean value in the vehicle control animals was 1672 DPM. The calculated stimulation indices (SI), representative of the change in 3H-thymidine incorporation in Tosyl chloride treated animals compared to controls, were 11.82, 23.13 and 17.38 for the tested doses of 5, 10 and 25% Tosyl chloride, respectively. Since the SI was >3 after 5% Tosyl chloride, the limiting value required for classification as a skin sensitiser, it was concluded that Tosyl chloride should be regarded as a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of Tosyl chloride (PTSC) was studied in mice. Three test groups of 4 female mice each were treated with 5, 10, and 25% w/v by means of open application of 25 μL to the dorsum of each ear (total 50 µL) for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H-thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a negative control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v).Group mean 3H-thymidine incorporations of 19763, 38675 and 29067 DPM were found in the auricular lymph nodes of animals treated with respectively 5, 10 and 25% Tosyl chloride. The group mean value in the vehicle control animals was 1672 DPM.The calculated stimulation indices (SI), representative of the change in 3H-thymidine incorporation in PTSC treated animals compared to controls, were 11.82, 23.13 and 17.38 for the tested doses of 5, 10 and 25% Tosyl chloride, respectively. Since the SI was >3 after 5% Tosyl chloride, the limiting value required for classification as a skin sensitiser, it was concluded that Tosyl chloride should be regarded as a skin sensitiser.

From this study it can be remarked that the data show a very high variability; there actually only two PTSC treated animals that have a higher DPM value than the control animal with the highest value. Because of the presence of non-responders (in dose groups) and high-responders (in control) it is difficult to proceed for classification on the basis of only two SI-values with dismissing the third concentration as it does not fit the hypothesis. For adequate distinction in classification between Cat.1A en 1B for GHS additional studies are needed.

 

Profiling of PTSC in OECD QSAR Toolbox indicates that PTSC can be expected to be reactive to protein. The results of the LLNA study are in agreement to this.

In the presence of water, PTSC is quickly hydrolysed to the acid Toluene-4-sulphonic acid (PTSAcid) and HCl. Evaluation of PTSAcid for sensitising properties in a Guinea pig Maximisation Test has shown that this substance is not sensitising.

In practice the use of PTSC has not indicated a great concern for sensitisation. From literature there is only one case reported of dermal sensitisation in a chemist working in a lab of a major pharmaceutical company.(Watsky KL et al., 1995, Occupational contact dermatitis from tosyl chloride in a chemist. Contact Dermatitis.29(4):211-2.)

Migrated from Short description of key information:

Tosyl chloride was found sensitising in the LLNA test when dermally applied at concentrations of 5% and higher.

Justification for selection of skin sensitisation endpoint:

Only available study

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

When a substance has been proven to be sensitising to skin, it is in principle also possible it could act as a sensitising substance by inhalation. In the case of PTSC there are two aspects that can be considered:

- The vp is 0.13 Pa at 20°C, and the respirable fraction (≤ 4 µm) of the crystalline solid is below 0,04%. Also likelihood of exposures by aerosols from the use of the substance is low (also taking into consideration the low water solubility of 3.1 g/L). Consequently the likelihood of exposures via inhalation is low.

- When PTSC get into contact to the mucous of the upper bronchi it will readily hydrolyse to toluene sulfonic acid and HCL, both strong corrosive acids, but not sensitising substances.

Migrated from Short description of key information:

No data available. Likelihood of exposure is very low.

Justification for classification or non-classification

The available data indicate a need for classification for Tosyl chloride based on the positive outcome in a LLNA which is consistent with its chemical profile indicating a possibility to react with protein. The available data is not suitable for sub-categorisation. The substance therefore is classified Cat.1 for skin sensitisation for GHS with H317: May cause an allergic skin reaction.

There is no information available that would indicate a need for classification for respiratory sensitisation.