Benzenesulfonic acid, 4-(branched alkyl derivs.) and benzenesulfonic acid, 4-(linear alkyl dervis.), calcium salts

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

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

Adequacy of study:

key study

Study period:

July and August 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study is similar to early test guidelines in compliance with GLP but without range finding data or concurrent assessment for irritation. As this data is used in a read-across approach Klimisch 2 is assigned.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca/Ola/Hsd strain

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Limited, Blackthorne, Bicester, Oxon, UK
- Housing: suspended stainless steel cages (18.4cm x 52.3cm x 12.7cm) with one solid sheet side and mesh front, floor, rear and remaining side
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: minimum of four days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 21±2°C
- Humidity (%): approximately 55±15%
- Air changes (per hr): approximately 25-30 air-changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

dimethylformamide

Concentration:

0.1, 1, 10 or 30% w/v

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS: none reported
MAIN STUDY
- Test method: Approximately 25 µL of a 0.1, 1, 10 or 30% w/v concentration of the test sample in DMF was applied, using a variable volume micro pipette, to the dorsal surface of each ear, on three consecutive days .
- Criteria used to consider a positive response: one or more concentrations of the test chemical should elicit a 3-fold or greater increase in isotope incorporation relative to the vehicle control group.

Positive control substance(s):

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

Positive control results:

The sensitization potential of hexylcinnamaldehyde was assessed using the method described above. A vehicle control group was similarly treated using acetone.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The application of test material, at concentrations of 0.1, 1, 10 and 30% w/v in DMF, resulted in an increase in isotope incorporation which was greater than or equal to 3-fold at all four concentrations.

concentration (% w/v)	number of lymphnodes assayed	counts per minute (cpm)	cpm per lymphnode(x102)	test: control ratio
0 (vehicle only) 0.1 1    10 30	8    8   8   8 8	511    1904   1539   5200   10463	0.64    2.38   1.92   6.50   13.08	N/A    3.72   3.00   10.16   20.44

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The substance is considered sensitising to skin

Executive summary:

The substance (Total Base Number = 13) was assessed using the LLNA study method in mice tested at 0.1, 1, 10 and 30% w/v in DMF. All concentrations achieved greater or equal to to 3 -fold increase in isotope incorporation conpared to solvent control and the substance is considered sensitising at all concenrtrations.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Classification and Labelling Proposal for Benzenesulfonic acid, 4 -(mono-C15-36 branched alkyl derivs., C24 rich), and benzenesulfonic acid, 4-octyadecyl, calcium (EC No. 939-141-6) Classification and Labeling is proposed and explained for Benzenesulfonic acid, 4 -(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts using data from skin sensitisation studies on natural and synthetic calcium sulfonates. 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 calcium sulfonate is used for natural and synthetic long-chain alkylbenzenesulfonic acids, calcium salts. Calcium sulfonates, which have surfactant properties, are used as detergents in a broad variety of lubricant applications. In some cases, excess calcium carbonate is added to the calcium sulfonates to add acid buffering capacity (commonly known as “overbasing”). Calcium sulfonates with a large excess of calcium carbonate are referred to as high overbased or high total base number (TBN) calcium sulfonates, whereas calcium sulfonates with small amounts of added calcium carbonate are called low overbased or low TBN calcium sulfonates. Animal studies show that 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. However, human repeat insult patch tests clearly show that high TBN overbased calcium sulfonates (TBN ≥ 300) are not sensitisers and that low TBN calcium sulfonates do not cause sensitization in a substantial number of persons at concentrations of 10% or lower within the definition of sensitisation under EU Regulation (EC) No. 1272/2008. Calcium Sulfonate Skin Sensitisation: Low TBN Calcium Sulfonates In a 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 939-141-9; 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 an this low TBN substance ( Benzenesulfonic acid, 4-(mono-C15-36 branched alkyl derivs., C24 rich) and benzenesulfonic acid, 4-octadecyl, calcium salts; EC 939-141-9; TBN = 13) was evaluated in guinea pigs (Bonnette, 1993b; Table 30). 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. Table30. 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	Conclusion
		24 hours	24 hours
Test	50%	1.9	2.1	9/10	Sensitizer
Control	50%	0.2	0.2
Test	25%	1.6	2.0	10/10	Sensitizer
Control	25%	0.7	0.6
Test	10%	1.5	1.5	9/10	Sensitizer
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 (Benezenesulfonic 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 31). 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 of the six subjects suspective 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 31). 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 31. 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 32). 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 32). 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 32). 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 32). 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 32). 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 32). 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 32). 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 32). 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 32). 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 32. 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 deminishing @ 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 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 33). 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 33. Dermal Sensitisation Study (Modified Buehler Design) in Guinea Pigs with a 100% High TBNCalcium Sulfonate (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) Induction: Challenge Results (Kiplinger, 1992a)

Group	Concentration	Mean Draize Score		# Positive	Conclusion
		24 hours	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 similar to 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, 1994f; Table 34). 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 34). 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 34). 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 34. Human Repeat Insult Patch Tests (HRIPT) with a High TBN Sulfonic acids, petroleum, calcium salts; 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 35). 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 35). 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 35). 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 35). 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 35. 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%	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, 1994b
Challenge	1%	0/49	0/49

In accordance with EU CLP Regulation (EC) No. 1272/2008, classification is not required for high TBN calcium sulfonates (TBN ≥ 300). Conclusion The weight-of-evidence indicates that low TBN calcium sulfonates (TBN < 300) are skin sensitisers with a specific concentration limit (SCL) of 10% and that high TBN calcium sulfonates (TBN ≥ 300) are not skin sensitisers. 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 calcium sulfonates (TBN < 300) with a specific concentration limit of 10% and classification is not required for high TBN calcium sulfonates (TBN ≥ 300).