N-ethyl-o(or p)-toluenesulphonamide

Endpoint summary

Description of key information

Local Lymph Node Assay (LLNA) (OECDTG 429): non-sensitizing (read across from p-Toluenesulfonamide CAS 70-55-3)

Guinea pig maximization test (GPMT) (OECDTG 406): non-sensitizing (read across from N-Ethyltoluene-2-sulfonamide CAS 10-77-56-1)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Information is derived from an analogue

Justification for type of information:

The full read across justification report is attached under "Attached justification".

Summary
Within REACH, the obligation to conduct tests with vertebrate animals should be considered as a last resort, only after exhausting all potential sources of information on the physical and (eco)toxicological properties of chemicals [1].
Article 13 of REACH requires that use must be made whenever possible by alternatives to vertebrate animal tests, through the use of alternative methods as in vitro methods, (Q)SARs or from structurally related substances via grouping or read-across.
Annex XI of REACH offers the option to evaluate specific endpoints by read-across. According to Chapter R.6 (QSARs and grouping of chemicals) of REACH technical guidance documents (TGD), read-across can be applied for “substances whose physico-chemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity”. Application of the read-across concept requires that physico-chemical properties, human health effects and environmental effects or environmental fate may be predicted from data from a reference substance to make a prediction of the endpoint for the target chemical. This avoids the need to test every substance for every endpoint. This report provides the justification to use a cross-reading approach between Toluenesulfonamides (p-Toluenesulfonamide (p-TSA, CAS 70-55-3), o-Toluenesulfonamide (o-TSA, CAS 88-19-7) and o/p-Toluenesulfonamide (o/p-TSA, CAS 1333 07 9)) and is presented according to the requirements stipulated in Chapter R.6 (QSARs and grouping of chemicals) of the REACH TGD for N-ethyl-o(or p)-toluenesulfonamide (NETSA, CAS 8047-99-2).

1. Regulation (EC) No 1907/2006 of the European Parliament and of the Council. Off. J. Eur. Union: 1–849

Reason / purpose for cross-reference:

read-across source

Positive control results:

The stimulation index of Hexyl Cinnamic Aldehyde (25% in vehicle v/v) was 4.42.

Key result

Parameter:

SI

Value:

1.23

Test group / Remarks:

Concentration (% w/v): 50

Remarks on result:

other: SI was < 3 / up to concentrations of 50% p-Toluenesulfonamide tested / no dose response relationship

Parameter:

SI

Value:

1.48

Test group / Remarks:

Concentration (% w/v): 25

Parameter:

SI

Value:

2.97

Test group / Remarks:

Concentration (% w/v): 10

Parameter:

SI

Value:

1

Test group / Remarks:

Concentration (% w/v): 0

Interpretation of results:

not sensitising

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates).

Conclusions:

Under the experimental conditions of this study, p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%. These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide.

Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of p-Toluenesulfonamide were studied in mice. Three test groups of 4 female mice each were treated with 10, 25, and 50% 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 4974, 2473 and 2063 DPM were found in the auricular lymph nodes of animals treated with respectively 10, 25, and 50% p-Toluenesulfonamide. 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 p-Toluenesulfonamide treated animals compared to controls, were 2.97, 1.48 and 1.23 for the tested doses of 10, 25, and 50% p-Toluenesulfonamide, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of p-Toluenesulfonamide tested and no dose response relationship was apparent it was concluded that p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%.

These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide (NETSA).

Reference 2

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 - 25.5 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, 10, 25, 50% 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. Based on known hazard data of p-Toluenesulfonamide it was to be expected that dermal application of a 50% (and lower) concentration should not cause systemic toxicity and/or excessive skin irritation. Therefore, concentrations of 50%, 25% and 10% were selected for this 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:
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 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.

Key result

Parameter:

SI

Value:

1.23

Test group / Remarks:

Concentration (% w/v): 50

Remarks on result:

other: SI was < 3 / up to concentrations of 50% p-Toluenesulfonamide tested / no dose response relationship

Parameter:

SI

Value:

1.48

Test group / Remarks:

Concentration (% w/v): 25

Parameter:

SI

Value:

2.97

Test group / Remarks:

Concentration (% w/v): 10

Parameter:

SI

Value:

1

Test group / Remarks:

Concentration (% w/v): 0

Cellular proliferation data / Observations:

SI was < 3 / up to concentrations of 50% p-Toluenesulfonamide tested / no dose response relationship

No signs of local irritation or clinical signs of systemic toxicity were observed in any of the animals during 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:

not sensitising

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates).

Conclusions:

Under the experimental conditions of this study, p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%. These results were used for read-across to N-Ethyl-o (or p)-toluenesulfonamide (NETSA).

Executive summary:

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of p-Toluenesulfonamide were studied in mice. Three test groups of 4 female mice each were treated with 10, 25, and 50% 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 4974, 2473 and 2063 DPM were found in the auricular lymph nodes of animals treated with respectively 10, 25, and 50% p-Toluenesulfonamide. 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 p-Toluenesulfonamide treated animals compared to controls, were 2.97, 1.48 and 1.23 for the tested doses of 10, 25, and 50% p-Toluenesulfonamide, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of p-Toluenesulfonamide tested and no dose response relationship was apparent it was concluded that p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%.

These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide (NETSA).

Reference 3

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Information is derived from an analogue

Justification for type of information:

The full read across justification report is attached under "Attached justification".

Summary
Within REACH, the obligation to conduct tests with vertebrate animals should be considered as a last resort, only after exhausting all potential sources of information on the physical and (eco)toxicological properties of chemicals [1].
Article 13 of REACH requires that use must be made whenever possible by alternatives to vertebrate animal tests, through the use of alternative methods as in vitro methods, (Q)SARs or from structurally related substances via grouping or read-across.
Annex XI of REACH offers the option to evaluate specific endpoints by read-across. According to Chapter R.6 (QSARs and grouping of chemicals) of REACH technical guidance documents (TGD), read-across can be applied for “substances whose physico-chemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity”. Application of the read-across concept requires that physico-chemical properties, human health effects and environmental effects or environmental fate may be predicted from data from a reference substance to make a prediction of the endpoint for the target chemical. This avoids the need to test every substance for every endpoint. This report provides the justification to use a cross-reading approach between Toluenesulfonamides (p-Toluenesulfonamide (p-TSA, CAS 70-55-3), o-Toluenesulfonamide (o-TSA, CAS 88-19-7) and o/p-Toluenesulfonamide (o/p-TSA, CAS 1333 07 9)) and is presented according to the requirements stipulated in Chapter R.6 (QSARs and grouping of chemicals) of the REACH TGD for N-ethyl-o(or p)-toluenesulfonamide (NETSA, CAS 8047-99-2).

1. Regulation (EC) No 1907/2006 of the European Parliament and of the Council. Off. J. Eur. Union: 1–849

Reason / purpose for cross-reference:

read-across source

Key result

Reading:

other: not specified

Group:

test chemical

Dose level:

not specified

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

other: not skin sensitisng

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates).

Conclusions:

One of the GPMT tests was performed with Ethyl toluene sulfonamide according to the Magnusson and Klingman method. Under the conditions of the test, it was concluded that Ethyl toluene sulfonamide was not skin sensitising. These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide.

Executive summary:

Enstein et al. (1997) developed a quantitative structure-toxicity relationship model (QSTR) for assessing dermal sensitization using guinea pig maximization test (GPMT) results.The QSTR models were derived from 315 evaluated chemicals.One of the GPMT tests was performed with Ethyl toluene sulfonamide according to the Magnusson and Klingman method. Under the conditions of the test, it was concluded that Ethyl toluene sulfonamide was not skin sensitising.

These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide (NETSA).

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Existing in vivo data were used. Published in 1997 and the GPMT was performed latest 1997.

Specific details on test material used for the study:

Ethyl toluene sulfonamide (CAS 1077-56-1)

Species:

guinea pig

Strain:

not specified

Sex:

not specified

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 250 - 500 g

ENVIRONMENTAL CONDITIONS
not specified

Route:

intradermal and epicutaneous

Vehicle:

not specified

Concentration / amount:

The highest to cause mild-to-moderate skin irritation

Day(s)/duration:

Ca. day 0 - 8

Adequacy of induction:

not specified

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

not specified

Concentration / amount:

the highest non-irritant dose

Day(s)/duration:

ca. day 20 - 22

Adequacy of challenge:

not specified

No. of animals per dose:

15- 20

Positive control substance(s):

not specified

Key result

Reading:

other: not specified

Group:

test chemical

Dose level:

not specified

Remarks on result:

no indication of skin sensitisation

The cross-validated specificity of the equations range between 81 and 91% and the sensitivity between 85 and 95% for the dataset. For an independent test set, specificity us 79% and sensitivity is 82%.

Interpretation of results:

other: not skin sensitisng

Remarks:

in accordance with EU CLP (EC 1272/2008 and its updates)

Conclusions:

One of the GPMT tests was performed with Ethyl toluene sulfonamide according to the Magnusson and Klingman method. Under the conditions of the test, it was concluded that Ethyl toluene sulfonamide was not skin sensitising.These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide.

Executive summary:

Enstein et al. (1997) developed a quantitative structure-toxicity relationship model (QSTR) for assessing dermal sensitization using guinea pig maximization test (GPMT) results.The QSTR models were derived from 315 evaluated chemicals.One of the GPMT tests was performed with Ethyl toluene sulfonamide according to the Magnusson and Klingman method. Under the conditions of the test, it was concluded that Ethyl toluene sulfonamide was not skin sensitising.

These results were used for read-across to N-ethyl-o (or p)-toluenesulfonamide (NETSA).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Local Lymph Node Assay (LLNA) (OECDTG 429):

In a GLP-compliant OECD Guideline 429 study, skin sensitisation properties of p-Toluenesulfonamide were studied in mice. Three test groups of 4 female mice each were treated with 10, 25, and 50% 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 4974, 2473 and 2063 DPM were found in the auricular lymph nodes of animals treated with respectively 10, 25, and 50% p-Toluenesulfonamide. 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 p-Toluenesulfonamide treated animals compared to controls, were 2.97, 1.48 and 1.23 for the tested doses of 10, 25, and 50% p-Toluenesulfonamide, respectively. Since the SI was < 3, the limiting value required for classification as a skin sensitiser, in response to all concentrations of p-Toluenesulfonamide tested and no dose response relationship was apparent it was concluded that p-Toluenesulfonamide did not have a skin sensitising potential when dermally applied up to concentrations of 50%. This result was used for read-across to N-ethyl-o/p-toluene sulphonamide (NETSA).

Guinea pig maximization test (GPMT) (OECDTG 406)

Enstein et al. (1997) developed a quantitative structure-toxicity relationship model (QSTR) for assessing dermal sensitization using guinea pig maximization test (GPMT) results.The QSTR models were derived from 315 evaluated chemicals.One of the GPMT tests was performed with Ethyl toluene sulfonamide according to the Magnusson and Klingman method. Under the conditions of the test, it was concluded that Ethyl toluene sulfonamide was not skin sensitising.This result is used for read-across to N-ethyl-o/p-toluenesulphonamide (NETSA).

Justification for classification or non-classification

Based on the information available via read-across, to substance N-Ethyl-o (or p)-toluenesulfonamide (NETSA) does not have to be classified as skin sensitizer in accordance with the criteria outlined in Annex 1 of the CLP Regulation (1272/2008/EC).