Dermal Irritation Assessment of Three Benzene Sulfonate Compounds

Abstract

Three benzene sulfonate compounds, benzene sulfonate, benzene meta-disulfonate, and para-phenol sulfonate, were reported to be present in groundwater sampled from residential wells near a former disposal site. Concentrations ranged from <0.005 mg/L to 474 mg/L. Acute dermal irritation studies were performed on rabbits for each of the three sulfonate compounds to determine if they had the potential to cause irritation to the skin of persons using this groundwater for bathing, showering, or other uses where skin would be exposed. The studies were performed by Toxikon Corporation (Bedford, MA) in accordance with the United States Environmental Protection Agency (USEPA)’s 1998 Health Effects Test Guidelines. At the highest concentration tested (5000 mg/L), all three sulfonate compounds were considered to be slight irritants, producing very slight to mild erythema (Draize Score 1). In all cases, the reactions were reversible. At 2000 mg/L, benzene meta-disulfonate and para-phenol sulfonate caused no irritation and were considered not to be irritants. At 2000 mg/L, only benzene sulfonate was considered to be a slight irritant, producing a mild erythema that was completely reversible within 24 hours. Benzene sulfonate was not considered an irritant at 1000 mg/L or at 500 mg/L. It is important to note that all three sulfonate compounds produced only a slight irritation at the highest concentration tested. None of the compounds produced a moderate to severe irritation response (i.e., severe erythema, edema). Furthermore, any irritation response observed at the highest concentrations tested was reversible within 72 hours. The only irritation response observed at the second highest dose was also reversible within 24 hours.

Three benzene sulfonate compounds, benzene sulfonate, benzene meta-disulfonate, and para-phenol sulfonate, were reportedly detected in groundwater samples taken from residential wells near a former disposal site. Questions were raised about the possibility that dermal contact with groundwater containing the compounds might cause skin irritation. A comprehensive review of the scientific literature (e.g., Lewis 1992, 1993; Verschueren 1996; search of Medline and Toxline databases) found no information on the skin irritation properties of any of the three sulfonates. One reference to the irritation potential of a related compound, pure benzene sulfonic acid, was found (Lewis 1992). This source cited Smyth et al. (1962), which summarized rabbit irritation test data on 300 compounds. According to this reference (op. cit.), pure benzene sulfonic acid is a skin and eye irritant to rabbits. This is not surprising for a compound that is applied as the pure acid. However, in dilute neutral solutions, the compound would be present as the benzene sulfonate anion, which may not be irritating.

A review of the toxicity of zinc phenol sulfonate, a compound that only differs from para-phenol sulfonate in the type of metal (zinc) used to produce the salt, found no acute or subchronic dermal toxicity (skin irritation in rabbits; skin sensitization in guinea pigs) when tested as dilute (2% to 16%) aqueous solutions or powders (Cosmetic Ingredient Review [CIR] Expert Panel 1986). Minimal skin irritation was observed when tested as the pure zinc sulfonate compound. Additionally, responses to dilute aqueous solutions (2.5%) or powders (2.5% to 5%) were negative for both skin irritation and sensitization when tested in human volunteers. For this study, it would also be expected that the skin irritation testing of the three sulfonates, applied as the pure compounds, would induce some irritation to the skin. However, the three sulfonates are reportedly present in groundwater as very dilute neutral solutions. Consequently, the toxicological issue of interest is whether dilute benzene sulfonate solutions are irritating to the skin.

Acute dermal irritation studies, therefore, were conducted on laboratory animals for aqueous solutions of the dissociable salt of each of the following sulfonate compounds:

• benzene sulfonate (also referred to as sodium benzene sulfonate);

• benzene meta-disulfonate (also referred to as 1,3-benzenedisulfonate disodium salt); and

• para-phenol sulfonate (also referred to as sodium 4-hydroxybenzene sulfonate).

  Dermal irritation was defined as the production of reversible inflammatory changes in the skin following application of the sulfonate compound. A test substance was considered corrosive if it destroyed the structure of intact skin or changed it irreversibly. The toxicology of the counter ion notwithstanding, it is assumed, in the present study, that any observed toxicity is attributable to the anion.

METHODS

Test Materials/Chemicals

Sodium benzene sulfonate (CAS no. 515-42-4) was purchased from Sigma-Aldrich (14,728–1; Lot no. DU 11227LN). Disodium benzene 1,3-disulfonate (CAS no. 831-59-4; Lot no. G6341A) and sodium 4-hydroxybenzenesulfonate dihydrate (sodium para-phenol sulfonate dihydrate; CAS no. 825-90-1; Lot no. G9439A) were purchased from ABCR GmbH & Co., Germany.

Quality Assurance/GLPs

Each study was conducted in accordance with experimental protocols approved by The Office of Prevention, Pesticides and Toxic Substances (OPPTS), United States Environmental Protection Agency (USEPA), Health Effects Test Guidelines: OPPTS 870.2500, Acute Dermal Irritation, 1998, and applicable laws and regulations including:

• Good Laboratory Practice Regulations (U.S. EPA 40 CFR, Part 792);

• Guide for Care and Use of Laboratory Animals (National Academy of Sciences [NAS] 1996);

• Public Health Service Policy on Humane Care and Use of Laboratory Animals, Health Research Extension Act of 1985 (Public Law 99-158 November 20, 1985, Reprinted 1996); and

• Department of Agriculture, Animal and Plant Health Inspection Service, 9 CFR Ch.1 (1/1/95 edition), Sub-chapter A-Animal Welfare.

Experimental Groups

Male and female New Zealand white rabbits (Oryctolagus cuniculus) were purchased from the Millbrook Rabbit Breeding Labs in Amherst, MA. These animals were chosen for study based upon their historical use in dermal safety studies and upon OPPTS guidelines (USEPA 1998). Healthy rabbits at least 10 weeks old (adult) were purchased, with weights ranging between 2.03 and 2.24 kg (weighed to the nearest 10 g). Animals were randomized when received from the vendor by the assignment of a unique identification number. The animals used in the studies had not been used in previous experiments and were observed to be free from any skin irritation, trauma, or adverse clinical signs prior to initiation of the studies. The numbers of animals used in these studies were based upon OPPTS guidelines (USEPA 1998). An untreated patch of dorsal skin on each animal served as its control.

Rabbits were housed individually in suspended stainless steel cages with noncontact hardwood chips as bedding. Laboratory room temperature was 68°F (±5°F), and animal room relative humidity was between 30% and 70% with 10 to 15 air exchanges per hour. A 12-hour light/dark cycle of full spectrum fluorescent lights was maintained. The laboratory and animal rooms were maintained as limited-access facilities. Animals were fed TEK 8630 Rabbit Diet (from Harlan Teklad, Madison, WI) and tap water, ad libitum.

For the studies with benzene meta-disulfonate and para-phenol sulfonate, three male and three female rabbits were tested in two dosage groups. For the study with benzene sulfonate, six male and six female rabbits were tested in four dosage groups.

Experimental Protocol

Dermal application sites were prepared by clipping the anterior dorsal (scapular) section of the rabbit’s trunk free of hair 24 hours before application of the test substance. Prior to clipping their hair, the animals were examined to ensure that their skin was free from irritation, trauma, or disease. The animals were weighed prior to application of the sulfonate compound.

A total of three animals were treated for each applied concentration of each sulfonate compound. For benzene meta-disulfonate and para-phenol sulfonate, two doses were applied: 5000 and 2000 mg/L. For benzene sulfonate, four doses were applied: 5000 mg/L, 2000 mg/L, 1000 mg/L, and 500 mg/L. All of the tests used a distilled water vehicle (United States Pharmacopeia [USP]) with application volumes of 0.5 ml of each stock dosing solution. Each dose level was tested in a separate group of animals.

The pH of the 5000 mg/L test substances ranged from 7.07 to 6.22 prior to dosing and after the 4-hour exposure. The pH of the 2000 mg/L test substances, measured prior to dosing and after the 4-hour exposure, ranged from 7.04 to 6.96. For benzene sulfonate, the pH of the 1000 mg/L dosage was 7.01 and 6.98 prior to dosing and after the 4-hour exposure, respectively. For the 5000 mg/L dosage, the pH was 7.00 and 7.02 prior to dosing and after the 4-hour exposure, respectively.

Each dose of test compound (0.5 ml of each concentration for each dose cohort) was applied to a small area of skin (approximately 6 cm²) and covered with four layers of gauze patch held in place with nonirritating tape. Because the sulfonate compounds were in liquid form, they were applied to the gauze before application to the skin. The patch was loosely held in contact with the skin using a semiocclusive dressing for the exposure period of 4 hours. Access by the animal to the patch was prevented, eliminating the potential for ingestion or inhalation of the test substance.

At the end of the 4-hour exposure period, the patches were removed and the skin wiped to remove any test substance still remaining. Animals were observed for signs of erythema and edema at 1, 24, 48, and 72 hours after patch removal. Observations of erythema or edema were scored according to the Draize Scale for Scoring Skin Reactions (USEPA 1998). This scale assesses irritation as follows: 0, no erythema or edema; 1, very slight erythema and/or edema (barely perceptible); 2, well-defined erythema and/or slight edema; 3, moderate to severe erythema or moderate edema, and 4, severe erythema and/or edema. Animals were weighed before exposure and at the end of the exposure period (day 3) and observed daily for the incidence of any clinical signs of toxicity (other than erythema and edema). Observations for these clinical signs included but were not limited to rigor, changes in respiration pattern, and convulsion.

RESULTS

Body Weights and Clinical Observations

All test animals survived for the duration of the study and all exhibited a gain in body weight. No overt signs of toxicity were seen in any of the animals during the course of the study (Table 1).

Benzene Sulfonate

At a dose of 5000 mg/L, very slight erythema (Draize Score 1) was observed in all three test animals 1 hour after removal of the gauze containing the benzene sulfonate solution (Table 2). At the 24-hour observation point, very slight irritation (Draize Score 1) was observed in one of the three test animals. At the 48-and 72-hour observation points, the irritation was reversed, and no irritation was observed in any of the animals. Edema was not observed at any time. Control skin showed no signs of irritation.

At 2000 mg/L, very slight erythema (Draize Score 1) was observed in two of the three test animals 1 hour after removal of the benzene sulfonate solution. The irritation was reversed by 24 hours. No erythema was observed in any test animals at this dosage at 24, 48 or 72 hours.

Benzene sulfonate was also applied at 1000 and 500 mg/L. No erythema or edema was observed at either concentration. Control skin showed no signs of irritation.

Benzene meta-Disulfonate

Two concentrations of benzene meta-disulfonate were tested in separate groups of animals: 5000 and 2000 mg/L. At 5000 mg/L, very slight (one animal, Draize Score 1) and well-defined erythema (two animals, Draize Score 2) were observed 1 hour after removal of the test substance (Table 2). At the 24-hour observation point, only two animals demonstrated very slight irritation. By 48 hours, very slight irritation remained on only one of the two test animals. By 72 hours, no irritation was observed on any animal.

No erythema or edema was observed in any test animals at 2000 mg/L. Control skin showed no signs of irritation.

para-Phenol Sulfonate

Two concentrations of para-phenol sulfonate, 5000 and 2000 mg/L, were tested in separate groups of animals. At 5000 mg/L, well-defined erythema (Draize score of 2) was observed in one of the three test animals 1 hour after removal of the test substance (Table 2). At the 24- and 48-hour observation points, very slight irritation (Draize Score of 1) was seen in this animal. By 72 hours, the irritation had been reversed. In the other two test animals, very slight irritation (Draize Score of 1) was observed 1 hour after removal of the test substance. By 24 hours, the irritation was reversed in these two animals.

No erythema or edema was observed in any test animals at 2000 mg/L. Control skin showed no signs of irritation.

Results Summary

At the highest dose level (5000 mg/L), all three sulfonate compounds produced either very slight or well-defined erythema. At the second highest dose level (2000 mg/L), only benzene sulfonate produced very slight erythema, which was reversible within 24 hours. Benzene sulfonate did not produce irritation at 500 or 1000 mg/L. In all cases, the irritation was completely reversible within 72 hours.

DISCUSSION

Based on these results, benzene meta-disulfonate and para-phenol sulfonate are considered slight irritants at a concentration of 5000 mg/L, but not at 2000 mg/L. Benzene sulfonate was a slight irritant at concentrations of 2000 and 5000 mg/L, but no response was observed at 1000 mg/L. Although these results represent the only known dermal study for these particular compounds in an aqueous solution, the observations are consistent with primary skin irritation studies that were performed for similar sulfonated compounds, para-toluene sulfonic acid and zinc phenolsulfonate.

Monsanto Company (1992) administered p-toluene sulfonic acid in white petroleum jelly to 10 human volunteers on sites on their arms or backs, placing 0.2 ml of test material on the skin and occluding with a patch for a 24-hour period. In eight of the volunteers, 6.25% p-toluene sulfonic acid (62,500 mg/L) was not irritating to the skin after a 24-hour dermal exposure. In two volunteers, a 24-hour dermal exposure to this was mildly irritating and caused slight to moderate redness, but no edema or other skin effects. This latter study showed that the application of even the pure sulfonate compounds produce only a mildly irritating response when tested at very high concentrations.

According to the National Toxicology Program (NTP) (1998), sodium salts of arylsulfonic acids, such as benzene sulfonates, are generally considered to be low in toxicity. Toxicology studies conducted by the NTP were identified for a related compound, sodium xylenesulfonate. This compound is present in shampoos at levels as high as 0.2% (2000 mg/L) and in cleaning products at levels as high as 10% (100,000 mg/L) (NTP 1998). NTP performed 17-day, 14-week, and 2-year dermal toxicity studies of solutions of sodium xylenesulfonate in both rats (F344/N) and mice (B6C3F1).

In the 17-day study, rats received dermal doses of 0.3 ml, and mice received 0.1 ml of 5, 15, 44, 133, and 400 mg/ml solutions in distilled water. No significant treatment related effects in the skin were observed in any animal groups.

In the 14-week study, rats received 0.3 ml, and mice received 0.1 ml of 5, 15, 44, 133, and 400 mg/ml solutions in 50% ethanol. The only clinical finding reported was a brown discoloration of the skin at the site of dosing in rats. In both rats and mice, minimal hyperplasia of the epidermis was stated to be treatment related in animals dosed using concentrations of 400 mg/ml. Chronic skin inflammation was noted in three male and three female control mice and in one male and one female animal and was not considered treatment related. No skin inflammation was reported in rats.

In the 2-year study, rats received daily dermal doses of 0.085 to 0.357 ml of 75, 150, and 300 mg/ml solutions of sodium xylenesulfonate in 50% ethanol, and mice received 0.046 to 0.128 ml of 75, 150, and 300 mg/mL solutions in 50% ethanol. Volumes were adjusted for the weights of the animals throughout the study to maintain doses of 60, 120, or 240 mg/kg for rats and 182, 364, or 727 mg/kg for mice. Minimal to mild hyperplasia of the epidermis occurred at low incidences in male rats in the 60, 120, and 240 mg/kg dose groups and at low incidences in female rats in the control and 120 and 240 mg/kg dose groups. NTP concluded that this effect might have been related to chemical administration. However, skin inflammation and ulceration, which were noted in female rats, “did not appear related to chemical administration, but more probably were the result of repeated hair clipping and vehicle administration.”

Minimal to mild hyperplasia of the epidermis occurred at low incidences in male mice in the control, and 364 and 727 mg/kg dose groups and at low incidences in female mice in the control and 182, 364, and 727 mg/kg dose groups. In males, this effect may have been related to chemical administration. However, skin inflammation and ulceration, which was noted in female mice, was not considered related to chemical administration, because there was no irritation in male mice. In female mice, chronic irritation was reported in one mouse in each dose group, but this same effect was also noted in four control animals.

In summary, the NTP (1998) studied the dermal toxicology of sodium xylenesulfonate in rats and mice, using concentrations of the sulfonate in 50% ethanol as high as 300 mg/ml (300,000 ppm or 0.3%). This concentration exceeds the solubility of sodium xylenesulfonate, and the dosing solution at the highest concentration was a suspension. However, even at these high concentrations, which are much higher than those evaluated in the present rabbit study, sodium xylenesulfonate was not irritating to the skin to rats or mice. In addition, the reported hyperplasia of the epidermis occurred using only the 400 mg/ml dose in the 14-week study and at 75 mg/ml or higher in the 2-year study.

Given the results of the study reported here (and supported by scientific studies on related compounds), human dermal exposure to groundwater containing benzene sulfonate, para-phenol sulfonate, or benzene meta-disulfonate compounds is unlikely to cause skin irritation to residents living near the former disposal site. The dermal exposure of the general community would be much lower than the concentrations that were irritating to rabbits. Lastly, although residents use water everyday in ways that may lead to dermal exposure (e.g., bathing, showering, and washing dishes), the duration of their skin exposures are much less than the 4 hours of continuous dermal exposures used in these irritation studies. Residents would also be expected to towel dry skin or household items that were exposed to well water containing any or all of the three benzene sulfonates, which would further reduce dermal exposure. These facts, combined with the knowledge that the irritation studies described above used animals that are generally considered to be more sensitive to dermal irritation that humans (ECVAM 1997), allow one to conclude that the human irritation risk to these compounds by the dermal route is negligible for aqueous solutions containing less than 1000 mg/L of benzene sulfonate and 2000 mg/L of para-phenol sulfonate or benzene meta-disulfonate.