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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:
2010-01-27 to 2010-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-documented guideline study compliant to GLP
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
effective April 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: NIH report, The Murine Local Lymph Node Assay: A Test Method for Assessing the Allergic Contact Dermatitis Potential of Chemicals/Compounds, NIH No. 99-4494, 1999
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Laboratories, Bar Harbor, ME
- Age at study initiation: Approx. 2-3 months
- Weight at study initiation: 18.3 - 23.0 g (first screen animals); 17.5 -18.4 g (second screen animals); The weight variation of the animals at study start did not exceed ± 20% of the mean body weight.
- Housing: 1/cage in suspended wire-bottom cages. Bedding was placed beneath the cages and changed at least three times per week
- Diet (e.g. ad libitum): Fresh PMI Rodent Chow (Diet #5001) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

Animals were nulliparous, non-pregnant and assigned to the test groups using a computer-based random number program.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled and continuously monitored
- Humidity (%): continuously monitored
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2010-02-03 resp. 2010-03-02 To: end of study
Vehicle:
dimethylformamide
Concentration:
10%, 25% and 50%
Due to an error in dosing the screen, a second screen with a fourth group of two animals dosed at 15% of the test article was conducted.
No. of animals per dose:
2 animals per group (Preliminary Dermal Irritation Screen)
5 animals per group (Main Test)
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Concentrations of 10-50 % were used
Three groups of each 2 mice were treated with increasing concentrations of the test compound (10%, 25% and 50%) in DMF (N,N-Dimethylformamide). Due to an error in dosing the screen, a second screen with a fourth group of two animals dosed at 15% of the test article was conducted. Treatment was made by topical application of the test article concentrations to the dorsum of each ear once daily for three consecutive days (at approximately the same time each day). The test substance was spread over the entire dorsal surface of the ear using a micropipette at 25 µl/ear.

Sample preparation:
1st Screen: 250 µl of the test article were added to 250 µl of dimethylformamide (DMF) to yield a 50% (v/v) solution (brown liquid). Additional dilutions were made in DMF.
2nd Screen: 150 µl of the test article were added to 850 µl of DMF to yield a 15% (v/v) solution (dark brown liquid).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animal were assigned randomly
- Criteria used to consider a positive response:
An increase in ear thickness of 25% indicates a positive dermal irritation response.
A stimulation index SI > 3 indicates a sensitizing response.

TREATMENT PREPARATION AND ADMINISTRATION:
The test article concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test article in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal-irritation. Concentrations of 2.5%, 5% and 10% were used, so 100 µl of the test article were added to 900 µl of DMF to yield a 10% (v/v) solution (brown liquid). Additional dilutions were made in DMF.
Five groups of CBA/J mice (5 animals per group) were treated by topical application of the test article concentrations, vehicle control or positive control in the same manner as in the screen. DMF was chosen as the vehicle, based on solubility. Historically, the DMF (N,N-Dimethylformamide) vehicle has produced consistent results with the positive control 25% HCA (alpha-Hexylcinnamaldehyde), as well as with other sensitizers and non-sensitizers.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical evaluation of the calculated SI values were made by Student's t-test, using GraphPad InStat™, c. 1992-2003, version 3.06, 32 bit for Windows. However, determination of test article dermal sensitization was based on the criterion that test articles that yield a SI > 3 are characterized as potential sensitizing substances.
Positive control results:
The positive control 25% HCA (alpha-Hexylcinnamaldehyde) gave positive results, i.e. an increase in ear thickness of 25% which indicates a positive dermal irritation response, and a Stimulation Index of 11.8 ± 3.9, which is greater than 3 and clearly indicates a sensitizing response.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Mean Stimulation Index SI ± Standard deviation Vehicle Control (DMF) = 1.0 ± 0.4 Positive Control (25% HCA) = 11.8 ± 3.9** 2.5% Test material solution = 3.9 ± 0.8* 5% Test material solution = 4.6 ± 2.3** 10% Test material solution = 5.5 ± 2.0** * = statistically significant (p<0.5) over control ** = statistically significant (p<0.01) over control
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable because no radioactive thymidine analog was used. Instead, incorporation of 5-bromo-2'-deoxy-uridine (BrdU) as thymidine analog in proliferating cells was measured and expressed as BrdU+ cells.

Table 1: Ear Thickness (mm)

Treatment

Mean Ear Thickness (mm)

Change (%)

Pre-Dosing (Day 1)

48h (Day 3)

End In-Life (Day 6)

Day 3 – Day 1

Day 6 – Day 1

DMF (Vehicle control)

0.20

0.19

0.20

-5.0%

0.0%

25% HCA (Positive Control)

0.20

0.23

0.25

15.0%

25.0%

2.5% Test Material Solution

0.19

0.21

0.21

10.5%

10.5%

5% Test Material Solution

0.19

0.22

0.22

15.8%

15.8%

10% Test Material Solution

0.20

0.22

0.22

5.0%

10.0%

 

 

Table 2: Mean Number of Proliferating Cells in the Lymph Nodes of each Treatment Group

Treatment

Vehicle

Mean Total # Cells in Node

% BrdU+ LNC

Lymphocyte Proliferation (#BrdU+)

DMF (Vehicle control)

-

2281817

0.75

17441

25% HCA (Positive Control)

DMF

10358350

1.99

205535

2.5% Test Material Solution

DMF

5144250

1.35

67738

5% Test Material Solution

DMF

5479200

1.47

79667

10% Test Material Solution

DMF

5824467

1.67

95584

 

 

Table 3: Statistical analysis

Comparison of SI values by Student´s t-Test

Mean Difference

P Value

DMF (Vehicle control) vs. 2.5% Test Material Solution

-2.900

<0.05 *

DMF (Vehicle control) vs. 5% Test Material Solution

-3.560

<0.01 **

DMF (Vehicle control) vs. 10% Test Material Solution

-4.480

<0.01 **

 * = statistically significant (p<0.5)

** = statistically significant (p<0.01)

No differences regarding body weight / body weight gain and clinical observations compared to control were observed.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The skin sensitizing potential of the test item was determined in a well-documented guideline study compliant to GLP. Therefore, it can be considered as reliable without restrictions (Reliability 1) and the obtained results can be used for classification.
The topical application of the test material at 2.5%, 5%, and 10% resulted in SI values greater than 3 (SI > 3.0). Therefore, this test material is a dermal sensitizer in the Local Lymph Node Assay and does therefore need to be classified as Sensitizer - Skin Cat. 1.
Executive summary:

In a Local Lymph Node Assay according to EPA OPPTS 870.2600, OECD 429 and NIH No. 99-4494 with Benzenesulfonic acid, di-C10-14-alkyl derivs., calcium salts, 2,5%, 5%, 10% in N,N-Dimethylformamide (DMF), young adult female CBA/J mice (5 per dosage group) were tested. The sensitizing potential of the test item was determined by comparing the stimulation indices of both vehicle and treatment groups based on the relative amount of proliferating cells as determined via flow cytometry / 5-bromo-2’-deoxy-uridine (BrdU) incorporation. A 25% solution of alpha-Hexylcinnamaldehyde in DMF served as positive control. No systemic clinical effects and mortality were observed, whereas both positive control and test item induced a swelling of the ear, but no excessive local dermal irritation is indicated.

All test substance concentrations resulted in a Stimulation Index >3. So, in this study Benzenesulfonic acid, di-C10-14-alkyl derivs., calcium salts is a dermal sensitizer and needs to be classified as Sensitizer - Skin Cat. 1.

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

Classification and Labelling Proposal - Benzenesulfonic acid, di-C10-14-alkyl derivs., calcium salts - EC No. None - CAS No. None

 

Classification and Labeling is proposed and explained for Benzenesulfonic acid, di-C10-14-alkyl derivs., calcium salts using data from skin sensitization 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, calcium sulfonate is a common name 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 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 skin sensitization response. However, human repeat insult patch tests clearly show that high TBN overbased calcium sulfonates (TBN = 300) are not sensitizers 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 sensitization under EU Regulation (EC) No. 1272/2008.

 

Sensitisation:

 

Low TBN Calcium Sulfonates

 

In a key study the dermal sensitization of this substance (Benzenesulfonic acid, di-C10-14-alkyl derivs., calcium salts; EC (None); TBN = 3.9) was evaluated in the local lymph node assay (LLNA) (Carathers, M., 2010). Groups of female mice (5/group) were dose with approximately 25 µl of 2.5, 5, or 10% concentrations of the test material in DMF on the dorsal surface of each ear for three consecutive days. The animals were then injected with 5-bromo-2’-deoxy-uridine (BrdU), and the auricular lymph nodes were then collected and prepared for flow cytometry counting. A three-fold or greater increase in BrdU incorporation was observed at all test concentrations. Although investigators have reported that LLNA over predicts the sensitization potential of surfactants (Basketer DA & Kimber I, 2011; Ball et al, 2011), the positive response in this study is supportive of the sensitization response for low TBN calcium sulfonates noted in other sensitization studies.

 

In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278 -7; TBN = 13) was evaluated in the local lymph node assay (LLNA) (Lees, D., 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 were 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 sensitization potential of surfactants (Basketer DA & Kimber I, 2011; Ball et al, 2011), the positive response in this study is supportive of the sensitization response for low TBN calcium sulfonates noted in other sensitization studies.

 

 In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts;EC 616-278-7; TBN = 13) was evaluated in guinea pigs (Bonnette, K.L., 1993b; Table 1). 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 sensitization 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 scoreswere 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 sensitizer.

Table 1. Dermal Sensitisation Study (Modified Buehler Design) in Guinea Pigs with a 100% Low TBN Calcium Sulfonate (Benzene, Polypropene Derivs., Sulfonated, Calcium Salts; EC 616-278-7; TBN = 13) Induction: Challenge Results (Bonnette, 1993b)

Group

Concentration

Mean Draize Score

# Positive

Conclusion

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 naïve animals to account for primary irritation reactions

 In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in guinea pigs (Shults, S.K., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN =13) was evaluated in a human repeat insult patch test (Shanahan, R.W. & Erianne, J.A., 1993a; Table 2). 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 sensitizer.

 

In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in a human repeat insult patch test (Shanahan, R.W. & Erianne, J.A., 1994c; Table 2). 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 sensitizer.

 

Table 2:. Human Repeat Insult Patch Tests (HRIPT) with a Low TBN Calcium Sulfonate (Benzene, Polypropene Derivs., Sulfonated, Calcium Salts; EC 616-278-7; TBN = 13): Challenge Results

Test Phase

Concentration

# Positive Responses

Conclusion

Study

24 hours

72 hours

Induction

100%

 

 

Sensitiser

Shanahan & Erianne, 1993a

Challenge

100%

9/44

6/44

 

 

 

 

 

 

Induction

20%

 

 

Sensitiser

Shanahan & Erianne, 1994c

Challenge

20%

16/45*

16/45*

*48 or 96 hour 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 sensitizers. However, well-conducted, reliable, controlled HRIPT studies show that these substances do not cause sensitization in a substantial number of subjects at 10% and lower.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 30) was evaluated in a human repeat insult patch test (Alworth, K., Schwartz, H. & Erianne, J.A., 1995c; Table 3). 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 sensitization responses. Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions in all but one subject.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 30) was evaluated in a human repeat insult patch test (Eisenberg, R.R.,1994c; Table 3). 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 sensitization responses following the 10% challenge. Seven of the subjects that had positive responses during the challenge phase reported for rechallenge with a 1% concentration of the test substance. All except one subject had negative responses. The one subject exhibited a mild response (1+) only at the virgin site at the 24 hour observation. However, this site was noted as negative in the final (48 hour) evaluation. Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions at 10%.

 

In another key study the dermal sensitization of an analog 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, K., Schwartz, H. & Erianne, J.A., 1995a; Table 3). 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 sensitization responses. Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions.

 

In another key study the dermal sensitization of an analog 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, R.R.,1994a; Table 3). 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 sensitization responses following the 10% challenge.  Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions at 10%.

 

In another key study the dermal sensitization of an analog 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, K., Schwartz, H. & Erianne, J.A., 1995b; Table 3). 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 sensitization responses. Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions in all but one subject.

 

In another key study the dermal sensitization of an analog 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, R.R.,1994b; Table 3). 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 sensitization responses following the 10% challenge.  Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions at 10%

 

In another key study the dermal sensitization of an analog 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, R.W. & Erianne, J.A., 1994a; Table 3). 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 sensitization responses. Based on these results the test substance was determined to cause a low incidence of sensitization characterized by mild to moderate reactions in all but one subject.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 82), was evaluated in a human repeat insult patch test (Shanahan, R.W. & Erianne, J.A., 1994e; Table 3). 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 sensitization responses. Based on these results the test substance was determined to not cause a sensitization response.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 100) was evaluated in a human repeat insult patch test (Shanahan, R.W. & Erianne, J.A., 1994f; Table 3). 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 sensitization responses. Based on these results the test substance was determined to not cause a sensitization response.

 

In another key study the dermal sensitization of an analog of this substance substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 13) was evaluated in a human repeat insult patch test (DiFiglia, C.J., Shanahan, R.W. & Erianne, J.A., 1993b). 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 sensitization responses following the 1% challenge.  Based on these results the test substance was determined not to induce irritation or sensitization at 1%.

Table 3. Calcium Sulfonate Human Repeat Insult Patch Tests (HRIPT) with a 10% Induction Phase Concentration

Substance

Challenge

(%)

Challenge Response

Sensitisation 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 at 24 hours and severe erythema at 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 at original & virgin sites; 1/205 with moderate erythema at 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 at 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 616-278-7 (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 at 96 hrs with erythema deminishing at 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 616-278-7 (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

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

 

High TBN Calcium Sulfonates

 

In another key study the dermal sensitization of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) was evaluated in guinea pigs (Kiplinger, G.R., 1992a; Table 4). 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 sensitization 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 sensitizer.

Table 4. 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

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 sensitization of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 400) was evaluated in guinea pigs (Reagan, E.L., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 375) was evaluated in guinea pigs (Kiplinger, G.R., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in guinea pigs (Blaszcak, D.L., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in guinea pigs (Kiplinger, G.R., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of an analog of this substance (Benzene, polypropene derivs., sulfonated, calcium salts; EC 616-278-7; TBN = 300) was evaluated in guinea pigs (Bonnette, K.L., 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 sensitization 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 sensitizer.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (Shanahan, R.W. & Erianne, J.A., 1994d; Table 5). 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 sensitization responses following the 100% challenge.  Based on these results the test substance was determined not to induce irritation or sensitization at 100%.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (DiFiglia, C.J., Shanahan, R.W. & Erianne, J.A., 1993c; Table 5). 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 sensitization responses following the 10% challenge.  Based on these results the test substance was determined not to induce irritation or sensitization at 10%.

 

In another key study the dermal sensitization of an analog of this substance (Sulfonic acids, petroleum, calcium salts; EC 263-093-9; TBN = 300) was evaluated in a human repeat insult patch test (DiFiglia, C.J., Shanahan, R.W. & Erianne, J.A., 1993d; Table 5). 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 sensitization responses following the 1% challenge.  Based on these results the test substance was determined not to induce irritation or sensitization at 1%.

Table 5. 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%

0/200

0/200

 

 

 

 

 

 

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 sensitization of a high TBN analog 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, R.W. & Erianne, J.A., 1993b; Table 6). 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 sensitization.

 

In another key study the dermal sensitization of a high TBN analog 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, G.N., 1993; Table 6). 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 sensitization responses. Based on these results the test substance was determined to not cause a sensitization response.

 

 

In another key study the dermal sensitization of a high TBN analog 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, C.J., Shanahan, R.W. & Erianne, J.A., 1993a; Table 6). 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 sensitization responses. Based on these results the test substance was determined to not cause a sensitization response.

 

In another key study the dermal sensitization of a high TBN analog 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, R.W. & Erianne, J.A., 1994b; Table 6). 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 sensitization responses. Based on these results the test substance was determined to not cause a sensitization response.

Table 6. 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

0/511

 

 

 

 

 

 

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

148 hours

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 sensitizers with a specific concentration limit (SCL) of 10% and that high TBN calcium sulfonates (TBN = 300) are not skin sensitizers. Studies in guinea pigs and human volunteers show that low TBN benzene, polypropene derivs., sulfonated, calcium salts (EC 616-278-7; TBN = 13) are skin sensitizers. 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), and sulfonic acids, petroleum, calcium salts (EC 263-093-9; TBN = 30 to 100) show that low TBN calcium sulfonates do not cause sensitization 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 sensitization in guinea pigs. Results of guinea pigs studies at TBN = 300 are mixed; two studies ofsulfonic acids, petroleum, calcium salts, (EC 263-093-9) report no skin sensitization while one study of sulfonic acids, petroleum, calcium salts (EC 263-093-9) andone study ofbenzene, polypropene derivs., sulfonated, calcium salts (EC 616-278-7) report skin sensitization, However, numerous well-conducted, reliable, controlled human (HRIPT) studies with benzene, polypropene derivs., sulfonated, calcium salts ( EC 616-278-7; TBN = 300), sulfonic acids, petroleum, calcium salts (EC 263-093-9; TBN = 300), also show that high TBN (TBN = 300) do not cause skin sensitization. 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).


Justification for selection of skin sensitisation endpoint:
best study available

Justification for classification or non-classification

The weight-of-evidence indicates that the calcium sulfonate target substance (C10-C14) is a skin sensitiser (Cat. 1B, H317) with a specific concentration limit (SCL) of 10%, in accordance with CLP 1272/2008.

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