Registration Dossier

Administrative data

Description of key information

Discussion

Classification and Labelling Proposal for benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts(EC No. None; CAS No. None) - Classification and Labeling is proposed and explained for benzenesulfonic acid, di-C10-14-alkyl derivs., sodium saltsusing data from skin sensitisation studies with this substance and from skin sensitisation studies on natural and synthetic calcium sulfonates.

 

In a key LLNA BrdU ELISA study (OECD 442B), benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts [with a total base number (TBN) of 2.8] was tested on female CBA/J mice (five animals per group) were treated with test article concentrations of 5, 10, 25 and 50% in acetone:olive oil (4:1, AOO). A vehicle control group was treated with AOO and another group was treated with positive control, 25% alpha-hexylcinnamaldehyde in AOO were also included in the study design. Topical application of benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts resulted in SI values greater than 1.6 (SI > 1.6). Positive and vehicle controls were valid. Therefore, it was concluded that benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salt is a dermal sensitizer in the LLNA assay with a EC1.6 of 2.3% (v/v) (MB Research, 2018).

 

For Registration, Evaluation, Authorization and Restriction of Chemicals (REACH) classification and labeling, EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 applies. In the lubricant additive industry, a common name such as sodium and calcium sulfonate is used for natural and synthetic long-chain alkylbenzenesulfonic acids, sodium and calcium salts. Sodium and calcium sulfonates, which have surfactant properties, are used as detergents in a broad variety of lubricant applications. In some cases, excess sodium hydroxide or calcium carbonate is added to the calcium sulfonates to add acid buffering capacity (commonly known as “overbasing”). Sodium and calcium sulfonates with a large excess of sodium hydroxide or calcium carbonate are referred to as high overbased or high total base number (TBN) sodium and calcium sulfonates, whereas sodium or calcium sulfonates with small amounts of added sodium hydroxide or calcium carbonate are called low overbased or low TBN sodium and calcium sulfonates. Animal studies show that sodium and calcium sulfonates with a TBN greater than 300 are not skin sensitizers while the results in animals at a TBN of 300 exhibit a mixed response. Human repeat insult patch tests confirmed that the high TBN overbased calcium sulfonates (TBN ≥ 300) are not sensitisers and that low TBN calcium sulfonates do not cause sensitization (within the definition of sensitisation under EU Regulation (EC) No. 1272/2008) in a substantial number of persons at concentrations of 10% or lower. Based on this, a sensitisation specific concentration limit (SCL) of ≥ 10% for low TBN calcium sulfonates can be determined.

 

Sodium and Calcium Sulfonate Skin Sensitisation:

 

Low TBN Sodium and Calcium Sulfonates

In a dermal sensitisation of the low TBN substance (benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts; EC No. None; CAS No. None); TBN = 3) was evaluated in guinea pigs (Smedley, 2015a). The animals were treated topically with a 75% concentration of the test substance in mineral oil once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 35% concentration of the test substance in mineral oil. After approximately one week the animals were rechallenged with a 15% concentration of the test substance in mineral oil. At the respective 24 and 48-hours challenge and rechallenge readings, dermal scores of 2 were noted in 18/20 and 16/20 at the 35% challenge and 14/20 and 14/20 at the 15% rechallenge. The 24/48-hour Draize scores in the 35% challenge group were 1.9/1.8 compared to 0.6/0.6 in the challenge controls. The mean 24/48-hour Draize scores in the 15% rechallenge group were 1.7/1.7 compared to 0.7/0.8 in the rechallenge controls. Based on these results the test substance was determined to be a sensitiser.

In another key study the dermal sensitisation of a low TBN substance (benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-9; TBN = 13) was evaluated in the local lymph node assay (LLNA) (Lees, 1996). Groups of male mice (4/group) were dose with approximately 25 µL of 0.1, 1, 10 or 30% concentrations of the test material in DMF on the dorsal surface of each ear for three consecutive days. The animals were then injected with 3H-methylthymidine, and the auricular lymph nodes where then collected and prepared for scintillation counting. A three-fold or greater increase in isotope incorporation was observed at all test concentrations. Although investigators have reported that LLNA over predicts the sensitisation potential of surfactants (Basketer & Kimber, 2011; Ball et al., 2011), the positive response in this study is supportive of the sensitisation response for low TBN calcium sulfonates noted in other sensitisation studies.

 

In another key study the dermal sensitisation of a low TBN substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-9; TBN = 13) was evaluated in guinea pigs (Bonnette, 1993b; Table A). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 50% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. Following the first rechallenge, the animals then were rechallenged with a 10% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 50%, 25%, and 10% were 9/10, 10/10, and 9/10, respectively, and the mean 24/48-hour Draize scores were 1.9/2.1, 1.6/2.0, and 1.5/1.5, respectively. The mean 24/48-hour Draize scores for the naïve controls at 50%, 25%, and 10% were 0.2/0.2, 0.7/0.6, and 0.5/0.5, respectively. Based on these results the test substance was determined to be a sensitiser.

 

Table A. Dermal Sensitisation Study (Modified Buehler Design) in Guinea Pigs with a 100% Low TBN Calcium Sulfonate (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, Calcium Salts; EC 939 -141 -6; TBN = 13) Induction: Challenge Results (Bonnette, 1993b).

 

 Group

Concentration

Mean Draize Score

  # Positive

Conclusions

 24 hours

  48 hours

 Test

 50%

1.9 

2.1 

9/10 

Sensitiser

 Control

 50%

0.2 

0.2

 

 Test

 25%

1.6 

2.0

10/10 

Sensitiser

 Control

 25%

0.7 

0.6 

 

 Test

 10%

 1.5

 1.5

9/10 

Sensitiser

 Control

 10%

 0.5

 0.5

 

Control = Topical application of test article to naive animals to account for primary irritation reactions                

 

In another key study the dermal sensitisation of this low TBN substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939 -141 -6; TBN = 13) was evaluated in guinea pigs (Shults, 1993). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 25% concentration of the test substance. After approximately one week the animals were rechallenged with a 10% concentration of the test substance. Following the first rechallenge, the animals then were rechallenged with a 10% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 25%, 10% rechallenge, and 10% second rechallenge were 10/10, 9/10, and 7/10, respectively, and the mean 24/48-hour Draize scores were 1.4/1.15, 0.8/0.95, and 0.8/0.85, respectively. The mean 24/48-hour Draize scores for the naïve controls at 25%, 10% rechallenge, and 10% second rechallenge were 0.8/0.06, 0.55/0.6, and 0.3/0.65, respectively. Based on these results the test substance was determined to be a sensitiser.

 

In another key study the dermal sensitisation of this low TBN substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-6; TBN =13) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1993a; Table B). A panel of 53 subjects was identified for this test. In the induction phase the undiluted (100% concentration) test substance was administered directly to the skin and covered with a semi-occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 44 subjects that completed the induction phase was topically challenged with the undiluted (100% concentration) test substance. Positive responses (scores ≥ 1) were observed in 9/44 and 6/44 at the 24 and 72- hour readings, respectively. Rechallenge of the six subjects suspected of exhibiting allergic reactions confirmed allergic contact dermatitis in four (4/44 or 9%). Based on these results the test substance was determined to be a sensitiser.

 

In another key study the dermal sensitisation of this low TBN substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-6; TBN =13) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994c Table B). A panel of 55 subjects was identified for this test. In the induction phase a 20% concentration of the test substance in mineral oil was administered directly to the skin and covered with a semi-occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 45 subjects that completed the induction phase was topically challenged with a 20% concentration of the test substance in mineral oil. Positive responses (scores ≥ 1) were observed in 16/45 and 16/45 at the 24 and 72-hour readings (or 48 or 96-hour make up readings), respectively. Approximately 31 % (14/45) of the test population exhibited skin reactivity patterns that were suggestive or indicative of low to moderate grade, induced allergic contact dermatitis. Based on these results the test substance was determined to be a sensitiser.

 

Table B. Human Repeat Insult Patch Tests (HRIPT) with a Low TBN Calcium Sulfonate (Benzenesulfonic Acid, 4-(Mono-C15-36 Branched Alkyl Derivs., C24 Rich) and Benzenesulfonic Acid, 4-Octadecyl, Calcium Salts; EC 939 -141 -6; TBN = 13): Challenge Results

Test Phase

Concentration

# Positive Responses

Conclusion

Study

24 hours

72 hours

 Induction

 100%

 

 

Sensitizer

 

Shanahan & Erianne, 1993a

 Challenge

 100%

 9/44

 6/44

 Induction

 20%

 

 

Sensitizer

 

Shanahan & Erianne, 1994c

 Challenge

 20%

 16/45*

 16/45*

* 48 or 96 hr. make-up readings were required for some subjects

 

Low TBN Specific Concentration Limit (SCL)

The weight-of-evidence of all the animal and human studies demonstrates that low TBN calcium sulfonates are skin sensitisers. However, well-conducted, reliable, controlled HRIPT studies show that these substances do not cause sensitisation in a substantial number of subjects at 10% and lower.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 30) was evaluated in a human repeat insult patch test (Alworth, Schwartz & Erianne, 1995c; Table C). Five panels consisting of a total of 166 subjects were identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 142 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 8/142 (5.6%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions in all but one subject.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 30) was evaluated in a human repeat insult patch test (Eisenberg,1994c; Table C). A panel of 220 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of ten applications. Following a two-week rest period, each of the 205 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 12/205 (5.8%) of the subjects exhibited sensitisation responses following the 10% challenge. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions at 10%.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in a human repeat insult patch test (Alworth, Schwartz & Erianne, 1995a; Table C). Five panels consisting of a total of 159 subjects were identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 154 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 4/154 (2.6%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in a human repeat insult patch test (Eisenberg,1994a; Table C). A panel of 223 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of ten applications. Following a two-week rest period, each of the 199 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 12/199 (6.0%) of the subjects exhibited sensitisation responses following the 10% challenge. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions at 10%.

 

In another key study the dermal sensitisation of this substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-6; TBN =13) was evaluated in a human repeat insult patch test (Alworth, Schwartz & Erianne, 1995b; Table C). Four panels consisting of a total of 157 subjects were identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 140 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 4/140 (2.9%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions in all but one subject.

 

In another key study, the dermal sensitisation of this substance (Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-6; TBN =13) was evaluated in a human repeat insult patch test (Eisenberg,1994b; Table C). A panel of 227 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of ten applications. Following a two-week rest period, each of the 199 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 12/199 (6.0%) of the subjects exhibited sensitisation responses following the 10% challenge. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions at 10%.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 85), was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994a; Table C). A panel of 53 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with a semi-occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 48 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 3/48 (6.3%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to cause a low incidence of sensitisation characterized by mild to moderate reactions in all but one subject.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 13, was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994e; Table C). A panel of 60 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with a semi-occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 56 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 0/56 (0.0%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to not cause a sensitisation response.

 

In another key study the dermal sensitisation of a low TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 100) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994f; Table C). A panel of 52 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with a semi-occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 51 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 0/51 (0.0%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to not cause a sensitisation response.

 

In another key study the dermal sensitisation of an analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in a human repeat insult patch test (DiFiglia, Shanahan & Erianne, 1993d). A panel of 53 subjects was identified for this test. In the induction phase a 1% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 48 subjects that completed the induction phase was topically challenged with a 1% concentration of the test substance in mineral oil. A total of 0/48 (0%) of the subjects exhibited sensitisation responses following the 1% challenge. Based on these results the test substance was determined not to induce irritation or sensitisation at 1%.

 

Table C. Calcium Sulfonate Human Repeat Insult Patch Tests (HRIPT) with a 10% Induction Phase Concentration

Substance

Challenge %

Challenge Response

Sensitization Response (%)

Conclusion

Study

EC 263-093-9 (TBN = 30)

 10%

 7/142 with mild to moderate erythema/oedema including mild to moderate papular reactions; 1/142 with moderate erythema @ 24 hours and severe erythema @ 72 hours

 8/142 (5.6)

 Sensitiser: low incidence with mild to moderate reactions in all but one of 8 subjects

 Alworth, Schwartz & Erianne, 1995c

EC 263-093-9 (TBN = 30)

 10%

 3/205 with mild erythema @ original & virgin sites; 1/205 with moderate erythema @ both areas; 7/205 with mild erythema original site; 1/205 with mild erythema virgin site

 12/205 (5.8)

 Sensitiser: low incidence with mild to moderate reactions

Eisenberg, 1994c 

EC 616-278-7 (TBN = 13)

 10%

 2/154 with mild to moderate erythema with moderate papular reactions; 2/154 with mild to marked erythema and with moderate oedema and moderate papular reactions in the induction phase that were considered sensitised and, therefore were not challenged

 4/154 (2.6)

 Sensitiser: low incidence with mild to moderate reactions

 Alworth, Schwartz & Erianne, 1995a

EC 616-278-7 (TBN = 13)

 10%

 6/199 with mild erythema @ original sites; 5/199 with moderate erythema original site; 1/199 with moderate erythema virgin site

 12/199 (6.0)

 Sensitiser: low incidence with mild to moderate reactions

 Eisenberg, 1994a

EC 939-141-6 (TBN = 13)

 10%

  3/140 with mild to moderate erythema with mild to moderate oedema and/or mild to severe papular reactions in some; 1/140 with moderate erythema with mild to severe papular reaction including an adverse reaction report of reaction spreading to back/neck @ 96 hrs. with erythema diminishing @ 192 hours but papular reaction spreading to the eyes

 4/140 (2.9)

 Sensitiser: low incidence with mild to moderate reactions in all but one of 4 subjects

 Alworth, Schwartz & Erianne, 1995b

EC 939-141-6 (TBN = 13)

 10%

 1/210 with mild erythema original and virgin site; 6/210 with BP to mild erythema original site; 5/210 moderate erythema original site; 1/210 with marked erythema original site; 1/210 with mild erythema virgin site; 1/210 with marked erythema original site

 15/210(7.1)

 Sensitiser: low incidence with mild to moderate reactions

 Eisenberg, 1994b

EC 616-278-7 (TBN = 85)

 10%

 2/48 with mild to moderate erythema and 1/48 with moderate oedema and papular that spread beyond contact site during induction were not challenged

 3/48(6.3)

  Sensitiser: low incidence with mild to moderate reactions

 Shanahan & Erianne, 1994a

EC 263-093-9 (TBN = 82)

 10%

 56/56 no reaction

 0/56(0)

 Not a sensitiser

 Shanahan & Erianne, 1994e

EC 263-093-9 (TBN = 100)

 10%

 51/51 no reaction

 0/51(0)

 Not a sensitiser

 Shanahan & Erianne, 1994f

EC 263-093-9 = Sulfonic acids, petroleum, calcium salts

EC 616-278-7 = Benzene, polypropene derivs., sulfonated, calcium salts

EC 939-141-6 =Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts             

 

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification of low TBN calcium sulfonates is required for sensitisation with a specific concentration limit of 10%.

 

High TBN Calcium Sulfonates

In another key study the dermal sensitisation of the high TBN substance benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (EC No. None; CAS No. None; TBN = 448) was evaluated in guinea pigs (Smedley, 2015b). The animals were treated topically with a 100% concentration of the test substance in mineral oil once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 75% concentration of the test substance in mineral oil. After approximately one week the animals were rechallenged with a 50% concentration of the test substance in mineral oil. Following a one-week rest period the animal received a second rechallenge at 25% of the substance in mineral oil. At the respective 24 and 48-hours challenge, rechallenge, and second rechallenge readings, dermal scores of 1 were noted in 3/20 and 6/20 at the 75% challenge; dermal scores of 1 or 2 were noted in 13/20 and 13/20 at the 50% rechallenge; and dermal scores of 1 were noted in 10/20 and 12/20 at the 25% second rechallenge. The 24/48-hour Draize scores in the 75% challenge group were 0.2/0.3 compared to 0.3/0.5 in the challenge controls. The mean 24/48-hour Draize scores in the 50% rechallenge group were 1.2/1.2 compared to 0.9/0.3 in the rechallenge controls. The mean 24/48-hour Draize scores in the 25% second rechallenge group were 0.6/0.7 compared to 0.5/0.7 in the rechallenge controls. Based on the weight-of-evidence of these results the test substance was determined not to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) was evaluated in guinea pigs (Kiplinger, 1992a; Table D). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 25% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 25% challenge and 25% rechallenge were 0/12 and 0/12, respectively, and the mean 24/48-hour Draize scores were 0.4/0.4 and 0.4/0.4, respectively. The mean 24/48-hour Draize scores for the naïve controls at 25% challenge and 25% rechallenge were 0.1/0.1 and 0.4/0.3, respectively. Based on these results the test substance was determined not to be a sensitiser.

  

Table D. Dermal Sensitisation Study (Modified Buehler Design) in Guinea Pigs with a 100% High TBN Calcium Sulfonate (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) Induction: Challenge Results (Kiplinger, 1992a)

 Group

 Concentration

Mean Draize Score

# Positive

Conclusion

  24hours

 48 hours

 Test

 25%

 0.4

 0.4

 0/12

 Not a sensitiser

 

 Control

 25%

 0.1 

 0.1 

 

 Test

 25%

 0.4

 0.4

 0/12

 Not a sensitiser

 Control

 25%

 0.4

 0.3

 

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 400) was evaluated in guinea pigs (Reagan, 1988). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 60% concentration of the test substance. After approximately one week the animals were rechallenged with a 60% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 60% challenge and 60% rechallenge were 4/10 and 0/10, respectively, and the mean 26/48-hour Draize score severity indices were 0.7/0.5 and 0.0/0.2, respectively. The mean 26/48-hour Draize score severity indices for the naïve controls at 60% challenge and 60% rechallenge were 0.5/0.3 and 0.0/0.0, respectively. Based on these results the test substance was determined not to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) was evaluated in guinea pigs (Kiplinger, 1992b). The animals were treated topically with a 30% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 25% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 25% challenge and 25% rechallenge was 0/12 and 0/11, respectively, and the mean 24/48 -hour Draize scores were 0.4/0.3 and 0.4/0.3, respectively. The mean 24/48-hour Draize scores for the naïve controls at 25% challenge and 25% rechallenge were 0.5/0.4 and 0.2/0.2, respectively. Based on these results the test substance was determined not to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in guinea pigs (Blaszcak, 1992). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 25% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 25% challenge and 25% rechallenge were 1/20 and 7/20, respectively, and the mean 24/48-hour Draize scores were 0.0/0.2 and 0.4/0.4, respectively. In the naïve controls 0/10 and 4/10 animals, respectively, exhibited positive irritation responses; the mean 24/48-hour Draize scores for the naïve controls at 25% challenge and 25% rechallenge were 0.0/0.0 and 0.5/0.4, respectively. Dermal responses to the corn oil vehicle alone were like the treated animals and naïve control. Based on these results the test substance was determined not to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in guinea pigs (Kiplinger, 1992c). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 25% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 25% challenge and 25% rechallenge were 4/12 and 5/12, respectively, and the mean 24/48-hour Draize scores were 0.7/0.6 and 0.6/0.5, respectively. The mean 24/48-hour Draize scores for the naïve controls at 25% challenge and 25% rechallenge were 0.3/0.4 and 0.3/0.5, respectively. Based on these results the test substance was determined to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in guinea pigs (Bonnette, 1993a). The animals were treated topically with a 100% concentration of the test substance once per week during the three-week induction phase. Following a two-week rest period, the animals were topically challenged with a 50% concentration of the test substance. After approximately one week the animals were rechallenged with a 25% concentration of the test substance. Following the first rechallenge, the animals then were rechallenged with a 10% concentration of the test substance. The number of animals exhibiting positive sensitisation responses for the test substance at 50%, 25%, and 10% were 8/10, 10/10, and 7/10, respectively, and the mean 24/48-hour Draize scores were 1.1/2.1, 1.6/1.7, and 1.2/1.2, respectively. The mean 24/48-hour Draize scores for the naïve controls at 50%, 25%, and 10% were 0.5/0.5, 0.7/0.8, and 0.3/0.5, respectively. Based on these results the test substance was determined to be a sensitiser.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994d; Table E). A panel of 213 subjects was identified for this test. In the induction phase a 100% concentration of the test substance was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 200 subjects that completed the induction phase was topically challenged with a 100% concentration of the test substance. A total of 0/200 (0%) of the subjects exhibited sensitisation responses following the 100% challenge. Based on these results the test substance was determined not to induce irritation or sensitisation at 100%.

 

In another key study the dermal sensitisation of a high analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (DiFiglia, Shanahan & Erianne, 1993c; Table E). A panel of 53 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 51 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 0/51 (0%) of the subjects exhibited sensitisation responses following the 10% challenge. Based on these results the test substance was determined not to induce irritation or sensitisation at 10%.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (DiFiglia, Shanahan & Erianne, 1993d; Table E). A panel of 53 subjects was identified for this test. In the induction phase a 1% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 50 subjects that completed the induction phase was topically challenged with a 1% concentration of the test substance in mineral oil. A total of 0/50 (0%) of the subjects exhibited sensitisation responses following the 1% challenge. Based on these results the test substance was determined not to induce irritation or sensitisation at 1%.

 

Table E. Human Repeat Insult Patch Tests (HRIPT) with a High TBN Sulfonic acid, petroleum, calcium salt; EC 263-093-9; TBN = 300): Challenge Results

 Test Phase

 Concentration

#Positive Responses

Conclusion

Study

24 hours

72 hours

 Induction

100%

 

 

 Not a sensitiser

 

 Shanahan & Erianne, 1994d

 Challenge

100%

1/200

1/200

 Induction

10%

 

 

 Not a sensitiser

 

 DiFiglia, Shanahan & Erianne, 1993c

 Challenge

10%

0/51

0/51

 Induction

1%

 

 

 Not a sensitiser

 

 DiFiglia, Shanahan & Erianne, 1993d

 Challenge

1%

0/50

0/50

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1993b; Table F). Five panels consisting of a total of 241 subjects were identified for this test. In the induction phase a 100% concentration of the test substance was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 222 subjects that completed the induction phase was topically challenged with a 100% concentration of the test substance in mineral oil. The test substance did not induce any clinically significant irritation contact dermatitis. Only 1/222 (0.0%) of the subjects exhibited a reaction which was of questionable substance-related clinical significance. Based on these results the test substance was determined to not cause sensitisation.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in a human repeat insult patch test (Wachs, 1993; Table F). A panel of 57 subjects was identified for this test. In the induction phase a 10% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of ten applications. Following a two-week rest period, each of the 51 subjects that completed the induction phase was topically challenged with a 10% concentration of the test substance in mineral oil. A total of 0/51 (0.0%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to not cause a sensitisation response.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in a human repeat insult patch test (DiFiglia, Shanahan & Erianne, 1993a; Table F). A panel of 53 subjects was identified for this test. In the induction phase a 1% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 49 subjects that completed the induction phase was topically challenged with a 1% concentration of the test substance in mineral oil. A total of 0/49 (0.0%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to not cause a sensitisation response.

 

In another key study the dermal sensitisation of a high TBN analogue of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in a human repeat insult patch test (Shanahan & Erianne, 1994e; Table F). A panel of 53 subjects was identified for this test. In the induction phase a 1% concentration of the test substance in mineral oil was administered directly to the skin and covered with an occlusive patch three times per week for a total of nine applications. Following a two-week rest period, each of the 49 subjects that completed the induction phase was topically challenged with a 1% concentration of the test substance in mineral oil. A total of 0/49 (0.0%) of the subjects exhibited sensitisation responses. Based on these results the test substance was determined to not cause a sensitisation response.

 

Table F. Human Repeat Insult Patch Tests (HRIPT) with a High TBN Calcium Sulfonate (Benzene, Polypropene Derivs., Sulfonated, Calcium Salts; EC 616-278-7; TBN = 300): Challenge Results

  Test Phase

  Concentration

#Positive Responses

Conclusion

Study

24 hours

72 hours

 Induction

100%

 

 

Not a sensitiser 

Shanahan & Erianne, 1993b

 Challenge

100%

1/222

1/222

 Induction

10%

 

 

Not a sensitiser 

Wachs, 1993

 Challenge

10%

0/51

0/51

 Induction

1%

 

 

Not a sensitiser 

DiFiglia, Shanahan & Erianne, 1993a

 Challenge

1%

0/49

0/49

 Induction

1%

 

 

Not a sensitiser 

Shanahan & Erianne, 1994e

 Challenge

1%

0/49

0/49

 

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification is not required for high TBN calcium and sodium sulfonates (TBN ≥ 300).

Conclusion

The weight-of-evidence indicates that low TBN sodium and calcium sulfonates (TBN < 300) are skin sensitisers with a specific concentration limit (SCL) of 10% and that high TBN sodium and calcium sulfonates (TBN ≥ 300) are not skin sensitisers. Studies in guinea pigs show that low TBN benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (EC No. None; CAS No. None; TBN = 3) is a skin sensitizer while benzenesulfonic acid, mono-C20-24 (even)-sec-alkyl derivs., para-, sodium salts (EC No. None; CAS No. None; TBN = 448) is not a skin sensitiser. Studies in guinea pigs and human volunteers show that low TBN benzenesulfonic acid, 4-(mono-C15 -36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts (EC 939-141-9; TBN = 13) are skin sensitisers. Numerous well-conducted, reliable, controlled human (HRIPT) studies with benzene, polypropene derivs., sulfonated, calcium salts (EC 616-278-7; TBN values ranging from 13 to 85), sulfonic acids, petroleum, calcium salts (EC 263-093-9; TBN = 30 to 100), and benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts (EC 939-141-6; TBN = 13) show that low TBN calcium sulfonates do not cause sensitisation in a substantial number of subjects at 10% and lower. High TBN calcium sulfonates, sulfonic acids, petroleum, calcium salts (EC 263-093-9; TBN = 375 and 400) do not cause skin sensitisation in guinea pigs. Results of guinea pigs studies at TBN = 300 are mixed; two studies of sulfonic acids, petroleum, calcium salts, (EC 263-093-9) report no skin sensitisation while one study of sulfonic acids, petroleum, calcium salts (EC 263-093-9) and one study of benzene, polypropene derivs., sulfonated, calcium salts (EC 616-278-7) report skin sensitisation, However, numerous well-conducted, reliable, controlled human (HRIPT) studies with benzene, polypropene derivs., sulfonated, calcium salts (EC 616-278-7; TBN = 300) and sulfonic acids, petroleum, calcium salts (EC 263-093-9; TBN = 300) also show that high TBN (TBN ≥ 300) do not cause skin sensitisation. In accordance with EU CLP Regulation (EC) No. 1272/2008, classification is required for low TBN sodium and calcium sulfonates (TBN < 300) with a specific concentration limit of 10% and classification is not required for high TBN calcium sulfonates (TBN ≥ 300).

 

Value used for CSA :adverse effect observed (sensitising)

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:
10 Oct 2017- 22 Nov 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
CBA
Remarks:
J strain
Sex:
male/female
Details on test animals and environmental conditions:
Female CBA/J mice were received and following an acclimation period of at least five days, thirty-seven nulliparous, non-pregnant female mice were assigned to the test groups without conscious bias. The Day 1 body weight range for the QIT animals was 17.7 - 24.0 grams. The Day 1 body weight range for the main test animals was 19.3 - 23.0 grams. The weight variation of the animals at study start did not exceed ± 20% of the mean body weight. The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.

The animals were identified by cage notation and indelible tail marks, and housed one per cage in suspended wire-bottom cages. Bedding was placed beneath the cages and changed at least three times per week. Fresh Rodent Chow and water were available ad libitum. The animal room, reserved exclusively for mice on acute tests, was temperature-controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free. Temperature and humidity were continuously monitored using automatic recording devices.
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Concentrations of 5%, 10%, 25%, and 50% (v/v) of the test article were chosen for the main Local Lymph Node study.Two positive control groups were also tested, alpha-hexylcinnamaldehyde in AOO (25% HCA) and 1-chloro-2,4-dinitrobenzene in AOO (0.25% DNCB)
No. of animals per dose:
The main test was conducted with seven separate groups of healthy female CBA/J mice (five animals per group)
Details on study design:
The test article was tested for solubility in acetone:olive oil (4:1, AOO) and found to be soluble at a concentration of 50% (v/v). A preliminary dermal irritation screen was conducted with three groups (two animals per group) of healthy female CBA/J mice to determine the concentrations of the test article to be used in the main Local Lymph Node study. Three concentrations of the test article were tested: 10%, 25% and 50% (v/v). The results of this preliminary screen indicated that the 50% (v/v) test article treatment resulted in significant dermal irritation. Based on these results, concentrations of 5%, 10%, 25%, and 50% (v/v) of the test article were chosen for the main Local Lymph Node study.

Dosing
Preliminary Dermal Irritation Screen: Three groups of CBA/J mice (two animals per group) were treated with increasing concentrations of R-9818 (10%, 25% and 50% [v/v]) in AOO. Treatment was made by topical application of the test article concentrations to the dorsum of each ear once daily for three consecutive days. The test article was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μl/ear.

Type and Frequency of Observations
All animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.

Measurements
Body weights were recorded on Day 1, immediately prior to dosing, and on Day 6 (prior to euthanasia).

Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before euthanasia (approximately 120 hours after the first dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.

Lymph Node Isolation and Processing
On Day 6 of the preliminary dermal irritation screen, each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made at euthanasia on Day 6. The lymph nodes were not collected. On Day 5 of the main test, approximately 96 hours following the initial dose, and approximately 24 hours prior to euthanasia, the mice were injected with BrdU, in Dulbecco's phosphate-buffered saline (DPBS) at a dose of 500 μl per mouse (10 mg/ml). The BrdU was administered by intraperitoneal injection using a 26-gauge needle. This thymidine analog becomes incorporated into the DNA of proliferating cells, including proliferating nodal lymphocytes. On Day 6, each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made, and the lymph nodes were collected. The auricular lymph nodes were combined for each animal and single-cell suspensions were generated in RPMI-10 medium.

Determination of BrdU Incorporation
A BrdU-specific Enzyme-Linked Immunosorbent Assay (ELISA) kit, Lot No. 24890800, was obtained from Roche Applied Science, Indianapolis, IN, on 29 Sep 2017 (expiration date Mar 2019). The kit was used to
measure the amount of BrdU incorporation in the LNC suspensions. An aliquot of 100 μl of the LNC preparation was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to bind. Excess unbound anti-BrdU antibody solution was then removed by washing and the substrate solution was added and allowed to
generate chromogen. Absorbance of each well was measured using a MicroQuant plate reader (Bio-Tek Instruments) at 370 nm with a reference wavelength of 492 nm. The mean optical density (OD) values of three “blank” wells
(containing PBS) were used as a reference. Calculations were performed using MS Excel as describe in Analysis of Stimulation Index Data.

Analysis of Stimulation Index Data
Calculation of Stimulation Index (SI): For each animal, lymph node cell proliferation was determined by BrdU-ELISA and the mean Optical Density (OD) and SD were then calculated for each group. The mean OD for each animal was then divided by the mean number of proliferating lymphocytes in the vehicle control group. This “Test / Control Ratio” is the “Stimulation Index” (SI) and was calculated for each
animal as follows:
Mean ODtest article/Mean ODvehicle = Stimulation Index (SI) per animal
The mean SI and S.D. were calculated for each group.

EC1.6 calculation
The concentration at which the test article would produce a positive sensitization response (EC1.6) could not be calculated because no concentration produced an SI value less than 1.6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: 1-chloro-2,4-dinitrobenzene in AOO (0.25% DNCB)
Statistics:
For each dose group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Students’ t-Test was run to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. The EC1.6 is the sole determinant for a positive sensitization response.

Positive control results:
Positive control results within historical laboratory data
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
1
Variability:
S.D.= 0.5
Test group / Remarks:
Vehicle Control
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.3
Variability:
S.D= 1.3
Test group / Remarks:
5% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.3
Variability:
S.D.= 0.6
Test group / Remarks:
10% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.8
Variability:
S.D= 0.8
Test group / Remarks:
25% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
4.3
Variability:
S.D.= 1.7
Test group / Remarks:
50% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.7
Variability:
S.D.= 1.6
Test group / Remarks:
25% HCA (Positive Control)
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
5.7
Variability:
S.D. = 1.4
Test group / Remarks:
0.25% DNCB (Positive Control)
Cellular proliferation data / Observations:
A preliminary dermal irritation screen was performed and ear thickness measurements were used to assess irritation potential. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application, and on Day 6 before euthanasia. The 50% (v/v) test article treatment resulted in increases in ear thickness of more than 25%, and therefore the test article was considered irritating at this concentration. Based on these screen results, test article concentrations of 5%, 10%, 25%, and 50% (v/v) were chosen for the main Local Lymph Node Assay. In the main test, the 10%, 25%, and 50% (v/v) test article treatments resulted in excessive dermal irritation. The 25% HCA positive control also had a greater than 25% increase in ear thickness on Day 6, indicating that 25% HCA is an irritant as well as a sensitizer, but the 0.25% DNCB positive control had no significant increase in ear thickness.
Stimulation Indices were calculated by dividing the mean optical density value (a representation of the total amount of LNC proliferation) for each animal by the mean optical density value for the vehicle control group. The mean SI and S.D. for each treatment group was then calculated. A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI of 1.6 or more.

Test Article Dosing
No difficulties were experienced with the application of the test article to the ears or with the retention of test article by the ear surface.

Body Weights
Body weight losses were noted but were not significant (less than 2 grams). See Table 1 for individual body weight data.

Mortality and Systemic Observations
All animals survived the in-life phase of the study. Prior to the study start, all animals were observed to ensure that no skin lesions were present on the ears

Erythema Scores
The 25% and 50% (v/v) test article treatments in the screen, and the 5%, 10%, 25%, and 50% test article treatments in the main test produced no more than very slight erythema (barely perceptible). The 25% HCA and 0.25% DNCB positive controls also produced no more than very slight erythema

Ear Thickness as a Measure of Dermal Irritation
A preliminary dermal irritation screen was performed and ear thickness measurements were used to assess irritation potential. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application, and on Day 6 before euthanasia. The 50% (v/v) test article treatment resulted in increases in ear thickness of more than 25%, and therefore the test article was considered irritating at this concentration. Based on these screen results, test article concentrations of 5%, 10%, 25%, and 50% (v/v) were chosen for the main Local Lymph Node Assay. In the main test, the 10%, 25%, and 50% (v/v) test article treatments resulted in excessive dermal irritation. The 25% HCA positive control also had a greater than 25% increase in ear thickness on Day 6, indicating that 25% HCA is an irritant as well as a sensitizer, but the 0.25% DNCB positive control had no significant increase in ear thickness.

Determination of the Stimulation Index (SI)
Stimulation Indices were calculated by dividing the mean optical density value (a representation of the total amount of LNC proliferation) for each animal by the mean optical density value for the vehicle control group. The mean SI and S.D. for each treatment group was then calculated. A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI of 1.6 or more.

EC1.6 calculation
The EC1.6 was estimated to be 2.3% (v/v).

The %B cells, %T cells, %I-Ak + cells, and %I-Aκ +CD69+ cells were determined by flow cytometry as described in the Experimental Design section of this report. B:T ratios were then calculated. Group means and standard deviations are tabulated below. Test groups that show an increase of 25% or more (≥1.25 fold) when compared to the vehicle control in these LNC surface markers and have an SI value of 1.6 or more are considered dermal sensitizers. All four concentrations of the test article had a positive response, indicating that this test article is a true sensitizer. The positive controls 25% HCA and 0.25% DNCB were also found to be sensitizing.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Topical application of test article R-9817 at 5%, 10%, 25%, and 50% (v/v) in AOO resulted in SI values greater than 1.6 (SI > 1.6). Therefore, this test article is a dermal sensitizer in the Local Lymph Node Assay. In addition, all four concentrations of the test article yielded a positive response when analyzed by immunophenotyping, confirming that this test article is a sensitizer. The EC1.6 value was estimated to be 2.3% (v/v)
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The classification and labelling as sensitiser for benzenesulfonic acid, di-C10-14-alkyl derivs., sodium salts (with a TBN of 2.8) is based on the LLNA positive results. In addition, the sensitisation weight of evidence for this category of sulfonates, is based on a series of studies conducted according to the Modified Buehler design and on the results of numerous skin sensitisation animal studies with low and high TBN calcium sulfonates. Results of Human Repeat Insult Test (HRIPT) with low and high TBN calcium sulfonates have also been considered. In accordance with EU CLP Regulation (EC) No. 1272/2008, classification is required for low TBN sodium sulfonates (TBN < 300) with a specific concentration limit of 10%, and classification is not required for high TBN sodium sulfonates (TBN ≥ 300).