Amended Safety Assessment of Naturally-Sourced Clays as Used in Cosmetics

Attapulgite

The structurally important element is the amphibole double silica chain oriented with its long direction parallel to the c-axis. ¹ Attapulgite consists of double silica chains situated parallel to the c-axis with the chains linked together through oxygens at their longitudinal edges. Tetrahedral apexes in successive chains point in the opposite direction. The linked chains form a kind of double-ribbed sheet with two rows of tetrahedral apexes at alternate intervals in the top and bottom of the sheets. The ribbed sheets are arranged so that the apex oxygens of successive sheets point together and are held together by aluminum and/or magnesium in octahedral coordination between the apex oxygens of successive sheets. Chains of water molecules run parallel to the c-axis and fill the interstices between the amphibole chains. Aluminum substitutions for silicon are considered probable.

Bentonite, Hectorite, and Montmorillonite

Bentonite, Hectorite, and Montmorillonite (also known as smectites) units comprise of two silica tetrahedral sheets with a central alumina octahedral sheet. ¹ All tetrahedral tips point in the same direction and toward the center of the unit. The tips of the tetrahedrons of each silica sheet and one of the hydroxyl layers of the octahedral sheet form a common layer. As in Kaolin, the atoms common to both the tetrahedral and octahedral layer are O instead of OH. These layers are continuous in the a and b directions and are stacked one above the other in the c direction. As a consequence, O layers in the units become adjacent and a very weak bond is created with the possibility of cleavage. The preeminent feature of these clay ingredients is the ability of water and organic molecules to enter between unit layers and expand in the c direction. Expansion properties are reversible; however, the structure is completely collapsed by removal of interlayer polar molecules. Most of these clay ingredients have substitutions within their lattices: aluminum or phosphorous for silicon in the tetrahedral coordination and/or magnesium, iron, zinc, nickel, lithium, etc. for aluminum in the octahedral sheet.

Illite

Illite is a non-expanding, clay-sized, dioctahedral mineral that is considered part of the mica group.^5,6 It is a layered alumino-silicate, known as a phyllosilicate. Its basic unit is a layer composed of two inward-pointing silica tetragonal sheets with a central alumina octahedral sheet. Poorly hydrated potassium cations occupy the space between the sequence of layers, which prevents swelling or the expansion of the layers. Illite is very similar structurally to common mica (muscovite), with slightly more silicon, magnesium, iron, and water, and slightly less tetrahedral aluminum and interlayer potassium.

Kaolin

Kaolin’s structure is composed of a single silica tetrahedral sheet and a single alumina octahedral sheet combined in a unit so that the tips of the silica tetrahedrons and one of the layers of the octahedral sheet form a common layer. ¹ All the tips of the silica tetrahedrons point in the same direction and toward the center of the unit made by the silica and octahedral sheets. Composite octahedral-tetrahedral layers are formed due to the similarity between the sheets a and b dimensions. The common layer between the octahedral and tetrahedral groups consists of two-thirds of shared atoms between silicon and aluminum that become O instead of OH. Analyses of Kaolin have shown there is little substitution within the lattice. In a small percentage of cases, iron and/or titanium has replaced aluminum. This has only been seen in the relatively poor crystalline varieties of Kaolin.

Attapulgite

Attapulgite is produced through an opencast mining technique, stripping layers with heavy machinery. ¹ The clay is then transported to a processing plant where crushing, drying, classification, and pulverizing take place. High-heat drying to remove water may occur to enhance absorbent qualities. Attapulgite is mined in 10 countries: Australia, China, France, India, Russia, Senegal, South Africa, Spain, Turkey, and the US.

Bentonite

Large deposits of Bentonite have been discovered in Canada, China, France, Germany, Great Britain, Greece, Hungary, Italy, Japan, Mexico, New Zealand, North Africa, Poland, South Africa, the post-Soviet states, and the US. ¹ The mine ore of Bentonite is processed to remove grit and non-swelling materials.

A supplier has reported that Bentonite is mined mineral. ¹² The material is then washed, filtered, dried, treated, and tested for quality.

Clay

A supplier has reported that Clay (75% Illite, 19% Kaolin, and 6% Montmorillonite) is naturally sourced, mechanically refined, and not chemically processed. ⁹ A dehydration process is used to eliminate bacteria prior to sorting through induction.

Illite

Illite is formed by the weathering of silicates (feldspar), by the alteration of other clay minerals, or during the degradation of muscovite.^5,6 The formation of Illite is generally favored by alkaline conditions and by high concentrations of aluminum and potassium. Deposits of Illite are widely distributed globally: it is commonly found in soil and argillaceous sedimentary rocks, as well as in some low-grade metamorphic rocks.^5,6,8

Kaolin

Deposits of Kaolin have been found in England, the US, France, the Czech Republic and Slovakia, Germany, and Japan. ¹ Kaolin is extracted from kaolinized granite by washing it out with powerful water hoses. The clay stream is then pumped to the separation plant where sand and mica are removed. The purified clay is filtered when wet and then dried. The very fine powder is formed by milling.

Attapulgite

Attapulgite commonly is found with smectites, amorphous silica, chert, and other minerals. ¹ A typical mineral composition of Attapulgite is approximately 55% silicon dioxide, 10% aluminum oxide, 3.5% iron (III) oxide, 10.5% magnesium oxide, 0.5% potassium oxide, and 20% water.

Bentonite

The principal constituent of Bentonite is Montmorillonite. ¹ However, other minerals such as Illite, kaolinite, and nonargillaceous detrital minerals can be present. Most bentonites appear relatively pure and other mineral contributions rarely exceed 10%. Cristobalite is often present. Montmorillonite compositions frequently vary either in its lattice structure or in the exchangeable ions present. A typical mineral composition of Bentonite is approximately 60% silicon dioxide, 20% aluminum oxide, 3% iron (II) oxide, 1.5% magnesium oxide, 0.6% calcium oxide, 0.6% potassium oxide, and 21% sodium oxide.

According to the Food Chemicals Codex, Bentonite is composed of natural smectite clays consisting primarily of colloidal hydrated aluminum silicates of the Montmorillonite or Hectorite type of minerals with varying quantities of alkalis, alkaline earths, and iron. ⁷ Food-grade Bentonite should contain no more than 5 mg/kg arsenic, no more than 15 mg/kg lead, no more than 1000 colony-forming units (cfu)/g aerobic microbes, and have no detectable Escherichia coli in a 25 g sample.

In particle size analysis of Bentonite, 90% of the particles were smaller than 68 µm, 50% were smaller than 5 µm, and 10% were smaller than 2 µm. ¹³ No particles were smaller than 0.9 µm. A second analysis of Bentonite showed that 90% of the particles were smaller than 25 µm, 50% were smaller than 6.5 µm, and 10% were smaller than 1.6 µm. ¹⁴ No particles were smaller than 0.5 µm.

Clay

A supplier has reported that Clay contains 75% Illite, 19% Kaolin, and 6% Montmorillonite and does not contain quartz. ⁹ Trace heavy metals may be present: <0.5 ppm antimony, <17 ppm arsenic, <0.5 ppm cadmium, <7 ppm cobalt, <0.5 ppm tin, <0.05 ppm mercury, <20 ppm nickel, and <20 ppm lead were detected using inductively coupled plasma-optical emission spectrometry (ICP-OES). Bacterial, yeast, and mold content were below the threshold of detection. Dioxin and polychlorinated biphenyl “results are near zero.”

Fuller’s Earth

Principal deposits of Fuller’s Earth include Montmorillonite, Bentonite, Attapulgite, and sepiolite. ¹

Hectorite

Principal impurities of Hectorite include calcite, dolomite, silica crystals, and grit. ¹ A typical mineral composition of Hectorite is approximately 56% silicon dioxide, 0.1% aluminum oxide, 0.03% iron (III) oxide, 25% magnesium oxide, 0.1% potassium oxide, 3% sodium oxide, 1% lithium dioxide, 6% fluorine, and 12% water.

According to some suppliers of Hectorite products (97–100% pure), crystalline silica (also described as quartz and/or CAS No. 14808-6-7) may be an impurity.^15,16 Quantities of crystalline silica were reported to be 1–3%.

Illite

The sheets of Illite are composed of silicon, magnesium, iron, aluminum, potassium, and water. ⁶ In analysis of 0.5 kg samples of 3 clay products containing Illite, the major element composition (as mean concentration) comprises silicon (23.02%), aluminum (8.80%), calcium (4.55%), iron (4.22%), potassium (3.19%), titanium (0.48%), sulfur (0.21%), phosphorus (0.10%), and manganese (0.041%). ¹⁷ Trace element impurities (as mean concentration) were identified as barium (426.70 mg/kg), rubidium (253.7 mg/kg), strontium (227.07 mg/kg), zinc (100.97 mg/kg), cesium (65.93 mg/kg), nickel (35.37 mg/kg), neodymium (30.23 mg/kg), lanthanum (28.00 mg/kg), lead (26.77 mg/kg), copper (25.07 mg/kg), arsenic (16.33 mg/kg), thorium (8.83 mg/kg), uranium (3.78 mg/kg), and bromine (<1 mg/kg). Bulk composition analysis indicated the samples contained calcite and quartz.

Kaolin

Quartz, mica, and feldspar are often found associated with Kaolin as the crude mineral and are often removed through screening and elutriation. ¹ Potentially pathogenic organisms were absent. The bacteria present were mostly gram-positive aerobic spore-formers. A typical mineral composition of Kaolin (reported as kaolinite) is approximately 45% silicon dioxide, 39% aluminum oxide, 0.8% iron (III) oxide, 0.08% magnesium oxide, 0.08% calcium oxide, 0.1% potassium oxide, 0.7% sodium oxide, 0.2% titanium (IV) oxide, and 14% water.

According to the Food Chemicals Codex, Kaolin is a purified clay consisting mainly of alumina, silica, and water. ⁷ Food-grade Kaolin should contain no more than 3 mg/kg arsenic and no more than 10 mg/kg lead.

According to some suppliers of Kaolin products (up to 100% pure), crystalline silica (described as quartz, free respirable silica, and/or CAS No. 14808-6-7) may be an impurity.^18,19 Quantities of crystalline silica were reported to be ≤2%.

Montmorillonite

A typical mineral composition of Montmorillonite is approximately 51% silicon dioxide, 20% aluminum oxide, 0.8% iron (III) oxide, 3% magnesium oxide, 2% calcium oxide, 0.1% potassium oxide, 0.04% sodium oxide, 0.1% zinc oxide, and 23% water. ¹

Attapulgite

Attapulgite is reported to be used in absorbents, pesticides, oil and petroleum treatment, and as a filler in many products. ¹

Bentonite

Bentonite is reported to be used in foundry sand bonding, bleaching clay in oil refining and decolorizers, filtering agents, water impedance, animal feed, pharmaceuticals, paint, plasticity increasers, and iron-ore pelletizing. ¹

Bentonite is generally recognized as safe (GRAS) as a direct food additive for humans (21 CFR§184.1155) and animals (21 CFR§582.1155). Bentonite is also GRAS as an indirect food additive in adhesives and components of coatings (21 CFR§175.105), in paper and paperboard components (as a colorant only, 21 CFR§176.170), in adjuvants as a colorant for polymers (21 CFR§178.3297).

Clay

Clay (natural) is GRAS as an indirect food additive in polymers (cellophane; 21 CFR§177.1200).

Fuller’s Earth

Fuller’s Earth is reported to be used as a military decontaminant for removal of hazardous materials from the skin. ³⁰

Hectorite

Hectorite has been approved for use in internally and externally applied products, as well as dentifrices and externally approved pharmaceuticals. ¹

Hectorite is reported to be used as drug-delivery system in anticancer therapy because of its biocompatibility, mechanical strength, and natural availability. ³¹

Illite

Illite has been studied for use in environmental remediation of contaminated soils and water as an adsorbent.^32-35 It also has been studied for use in veterinary applications, such as dietary supplements for swine ³⁶ and topical therapeutic treatment in equine injuries. ³⁷

Kaolin

Kaolin is reported to be used in the paper industry to fill and coat the surface of paper, as a filler in rubber and plastics, paint extender, ceramics manufacture, ink, adhesives, insecticides, medicines, food additives, bleaching, adsorbents, cement, fertilizers, crayons, pencils, detergents, porcelain enamels, paste, foundries, linoleum, floor tiles, and textiles. ¹ It has been classified by the National Formulary as a tablet and/or capsule diluent.

Kaolin clay is GRAS as an indirect food additive with no limitation other than current good manufacturing practice (21 CFR§186.1256). It is used in the manufacture of paper and paperboard that contact food. Kaolin (colloidal) is an approved over-the-counter (OTC) drug as an antidiarrheal active ingredient (21 CFR§335.10), an anorectal active ingredient (21 CFR§346.14), and a skin protectant active ingredient (from 4% to 20%; 21 CFR§347.10). Kaolin is used as a digestive aid (21 CFR§310.545); however, the data are currently inadequate to establish general recognition of the safety and effectiveness of this ingredient for this specified use. Kaolin is exempted from the requirement of a tolerance for pesticide residues when used on or in food commodities to aid in the control of insects, fungi, and bacteria (food/feed use; 40 CFR§180.1180).

Kaolin minerals (specifically kaolinite) have been studied for use in environmental remediation of contaminated soils and water.^34,35 This clay material has also been studied for use in veterinary applications, such as dietary supplements for swine ³⁶ and topical therapeutic treatment in equine injuries. ³⁷

Montmorillonite

Montmorillonite is reported to be used for food packaging and in paper manufacturing.^27,28 It also has been studied for use in environmental remediation of contaminated soils and water^34,35 and in veterinary applications, such as use in dietary supplements for swine. ³⁶

Clay

In ex vitro bioavailability studies using human skin models, the ability of transcutaneous passage of heavy metals (vanadium, lead, arsenic, barium, nickel, chromium, and aluminum) in 3 clay pastes was analyzed. ³⁸ The clay pastes were white Montmorillonite, Kaolin, and Clay (composed of 75% Illite, 19% Kaolin, and 6% Montmorillonite). Approximately 150 g of each product were tested with human skin samples in Franz cells and incubated for 24 h. The tested products, the diffusion liquids, and the storage liquids were then analyzed for metal content (details not provided). No detectable quantities of heavy metals were found in the diffusion or storage liquids. It was determined that the traces of heavy metal in the clay pastes did not penetrate cutaneous tissue.

Kaolin

In a dietary study, a group of 10 male Sprague-Dawley rats were fed a control diet plus 0.5 mL 20% Kaolin – 1% pectin for 48 h. ¹ Stool samples were collected 72 h later and analyzed for volume, sodium, potassium, and fat content. The results were a 103% increase in sodium, a 184% increase in potassium, and fat excretion remained at baseline.

Montmorillonite

Polydisperse and monodisperse [¹³⁴Cs]-fused Montmorillonite suspensions were administered to groups of 40 rats and mice and to 120 beagle dogs by a multiport nose-only inhalation exposure system. ¹ Aerosol concentrations ranged from 0.1 to 0.001 mg of fused Montmorillonite/L of air (additional properties not provided). Equal numbers of male and female rats and mice and 74 male and 46 female dogs were utilized. Single exposure times for rats and mice ranged from 25 to 45 min and for dogs 15 to 50 min. All animals were whole-body counted for the labeled particles. Five rats and 5 mice from each group were killed 4 h after exposure. The remaining rats and mice were killed at various times after exposure. Tissues from rats and mice were collected on post-exposure days 2, 4, 8, 16, 32, 64, 128, 256, 365, 512, 730, and 850. Tissues and excreta from the dogs were also collected on the same schedule, but also at 4, 5, 7, and 9 yr after inhalation exposure. Two dogs were scheduled for termination at times ranging from 4 h to 9 yr. All animals were necropsied and tissues from lungs, lung-associated lymph nodes, gastrointestinal tract, spleen, kidneys, abdominal lymph nodes, blood, skeleton, muscle, and skin were prepared for analysis of [¹³⁴Cs]-exposure. The mass of material deposited into the lungs of rats and mice was ∼0.01 to 0.1 mg and for dogs was ∼1 to 10 mg. The mass of Montmorillonite for all three species was <0.1 mg/g of lung. Clearance of the initial ¹³⁴Cs occurred by dissolution and mechanical clearance. Mechanical clearance from the nasopharynx was rapid, and the clearance rate was decreased to a negligible value for all three species within a few days. Most initial deposits were cleared via the gastrointestinal tract. Long-term mechanical clearance from the pulmonary region occurred at a constant rate for all species. Solubilization was the primary factor in long-term lung clearance for most particles inhaled by dogs and mechanical clearance was dominant in rats and mice. Most of the long-term clearance of deposited particles went to lung-associated lymph nodes in dogs and occurred at a slower rate as compared to rats and mice. Rats and mice had a rapid clearance from the pulmonary region, where most of the mechanical clearance occurred via the gastrointestinal tract. Long-term clearance of the particles in dogs occurred at 3500-d half-time in the lymph nodes and 6900-d half-time clearance in the gastrointestinal tract. The transport rate of the particles in the dog was 0.0002/d of the lung burden. The long-term biological clearance half-term day was 690 d for rats and 490 d for mice. The lymph node accumulation process was modeled by a short-term process that became negligible after a few days.

Radiolabeled, [¹³⁴Cs]-fused Montmorillonite particles were instilled into specific lung lobes or injected intraperitoneally into 32 beagle dogs. ¹ Necropsy was performed 34, 182, and 365 d later. Specific sites of instillation included right apical lobe, right cardiac lobe, right diaphragmatic lobe, right intermediate lobe, left apical lobe, left diaphragmatic lobe, and intraperitoneal. Initial burdens in the peritoneal cavity or the lungs ranged from 0.50 to 14 µCi for 29 dogs and from 42 to 64 µCi lung burdens for the other 3 dogs. Effective translocation half-time of lung instillations was 390 d. The accumulation rate of [¹³⁴Cs]-fused particles in the lymph nodes was 0.03% per day. Individual lung lobes cleared particles to 1–2 lymph nodes, and specific lymph nodes accumulated particles from 1 to 3 lung lobes. Lymph nodes that collected particles from the lung included the left mediastinal node and the left, left-middle, right, and right-middle tracheobronchial lymph nodes. The destination for translocated particles was primarily the nodes proximate to the tracheal bifurcation. Particles injected into the peritoneal cavity were translocated mainly to mesenteric lymph nodes and left and right sternal lymph nodes. A small percentage of particles went to the left tracheobronchial lymph node.

Dermal

Oral

Inhalation

Parenteral

Parenteral

Oral

Parenteral

Oral

Attapulgite

In a rat inhalation study, groups of 40 (20 male and 20 female) SPF Fischer rats were exposed to samples of Attapulgite dust mined in Lebrija or Leicester in inhalation chambers at a concentration of 10 mg/m³ for 6 h/d for 5 d/wk until they were killed (no further details were available). ¹ Negative and positive control groups received Kaolin and crocidolite, respectively, at 10 mg/m³. Four animals were killed at 3, 6, and 12 mo, and the remaining rats were allowed to live their life span. All animals were subject to necropsy; the lungs, liver, spleen, kidneys, and other relevant organs were examined microscopically. At microscopic examination, 1 peritoneal mesothelioma, 1 adenocarcinoma, and 3 bronchoalveolar hyperplasia were found in rats treated with Lebrija Attapulgite. Thirty-five rats had no proliferative changes. In rats treated with Leicester Attapulgite, proliferative lesions observed included 2 plural mesothelioma, 1 peritoneal mesothelioma, 1 malignant alveolar neoplasm, 2 benign alveolar neoplasms, and 8 bronchoalveolar hyperplasias. Twenty-seven rats had no proliferative lesions. Rats exposed to the negative-control Kaolin had 2 bronchoalveolar hyperplasias. Rats in the positive-control crocidolite group had 1 lung adenocarcinoma and 3 bronchoalveolar hyperplasias.

Kaolin

Kaolin was reported to be the negative control in the above rat inhalation study. ¹ The rats received 10 mg/m³ Kaolin for 6 h/d for 5 d/wk. Two bronchoalveolar hyperplasias were reported.

Attapulgite

In an intratracheal study, groups of 5 rats received a single instillation of Attapulgite at 1, 5, and 10 mg. ¹ One month after treatment, bronchoalveolar lavage and microscopic examination of the lungs were performed. The average length of the fibers was 0.8 µm, and 100% of the fibers were less than 3 µm. Every test animal had type A lesions, which are characterized by an accumulation of inflammatory cells, mostly macrophages, and epithelioid cells around fiber deposits. These inflammatory cells form a compact cellular infiltrate at the periphery of the deposits and some are focally dispersed throughout the alveolar region. The bronchoalveolar lavage (BAL) fluid had mostly macrophages and a small number of neutrophils at 5- and 10-mg doses. At the 5-mg dose, 3.6% of the cells were lymphocytes.

Two groups of 30 to 50 female Osbourne-Mendel rats received a single direct application to the left pleural surface by open thoracotomy of 40 mg of 1 of 2 Attapulgite samples. ¹ The samples were 90% pure with quartz being the other component. One dose consisted of fibers >4 µm and the other contained no fibers >4 µm. The rats were killed at the end of 2 yr. Pleural sarcomas were seen in 2/29 rats. The incidences of pleural sarcomas in the untreated groups were 3/491 and 17/615 of the rats receiving the pleural implants of Attapulgite. Of rats receiving the positive control, crocidolite, 14/29 developed pleural mesotheliomas.

Attapulgite (20 mg/mL of 0.9% sodium chloride) was injected into the pleural cavities of 36 Sprague-Dawley rats. ¹ The median fiber length was 0.77 µm. Two control groups, untreated and saline-injected, were utilized. Necropsy was performed after the rats died or killed when moribund. No mesothelial neoplasms were found in either controls or in rats treated with Attapulgite. Survival times between the Attapulgite-treated group and the controls were not statistically different.

Attapulgite was injected intrapleurally as a single dose of 0.5, 2, 4, 8, 16, or 32 mg into 6 groups of 25 Fischer 344 rats. ¹ Nearly all the fibers were <1 µm in length. Mesotheliomas were present in 2/140 treated rats compared to 1/79 incidences in control groups. The median life span was 839 days for Attapulgite-treated animals and 729 days for nontreated animals.

In another intrapleural study, injections of 20 mg of different Attapulgite fiber samples in 1 mL of saline were administered to 2-mo-old Sprague-Dawley rats. ¹ The control group received only a saline injection. All rats were allowed to live full life span. The mean length of Attapulgite fibers in this experiment was 0.77 µm. The number of groups were not reported; however, 36 rats were reported to comprise each group. Pulmonary and thoracic neoplasms were fixed and processed for histopathological examination. The survival time of the treated groups (788 ± 155 days) was very similar to that of the control groups (809 ± 110 days). The incidence of mesothelioma was 0% for control groups and treated groups. The researchers concluded Attapulgite was not carcinogenic in this study.

Samples of Attapulgite from Lebrija, Torrejon, and Leicester were injected intrapleurally as a single injection in groups of 20 male and 20 female SPF Fischer rats. ¹ Concentrations were not reported; however, fiber length was reported as < 2 µm, for Lebrija Attapulgite, at most 0.54% > 6 µm for Torrejon Attapulgite, and 19% > 6 µm for Leicester Attapulgite. Kaolin and saline were used as negative controls, and crocidolite was used as a positive control. The animals were allowed to live their life span but were killed if they appeared distressed. Upon death, necropsy and microscopic examination of tissue were performed. Dust extraction was obtained from granulomas removed from the diaphragm or mediastinal tissue. Mesotheliomas were reported in 2, 14, 30, and 34 rats for Lebrija Attapulgite, Torrejon Attapulgite, Leicester Attapulgite, and crocidolite, respectively. In the negative controls, no mesotheliomas were reported for the Kaolin and 1 mesothelioma was reported for the saline group. Lebrija Attapulgite dust extracted from the lung had fibers <2 µm. Material examined from Torrejon Attapulgite was fibrous and had fiber length up to 8 µm. Leicester Attapulgite fibers from extracted lungs were up to 25 µm. The investigators considered these fibers to be tumorigenic.

Three samples of 25 mg of Attapulgite dust were injected intraperitoneally into 40 Wistar rats. ¹ Electron microscopy of the sample revealed 37.5% of fibers <2 µm long and 70.0% < 5 µm. All animals were observed until they died either spontaneously or were killed. Saline was injected into 80 control animals. The time required to produce the first tumor in the rats was 257 days and the tumor incidence rate was 65%.

In a carcinogenicity study conducted with 3 samples of Attapulgite labeled Georgia, Lebrija, and Morimoiron, female Wistar rats (112, 115, and 114 per sample type, respectively) were injected intraperitoneally. ¹ Each sample was injected 1/wk for 9 wk at 60 mg per injection. Fiber dimensions for each of the samples Morimoiron, Georgia, and Lebrija were as follows: <50% fiber length was 0.7, 0.5, and 0.8 µm, respectively and <50% fiber diameter of 0.07, 0.07, and 0.04 µm, respectively. Some rats died spontaneously or others in poor health were killed. Surviving animals were killed 2.5 yr after treatment for necropsy. At necropsy, neoplasms or organs with suspected neoplasm tissue were fixed for microscopic examination. The percentage of rats with tumors were 3.5%, 3.5%, and 3.6% for the Morimoiron, Lebrija, and Georgia samples, respectively. These 3 samples were determined to be noncarcinogenic.

In another experiment by the same investigators, a fourth sample of Attapulgite from Caceres was tested in 30 rats. ¹ Intraperitoneal injections of 2, 4, and 4 mg were administered consecutively for 3 wk. The fiber length and diameter of this sample were <50% 1.3 and 0.07 µm, respectively. Animals in poor health were killed. Surviving animals were killed 2.5 years after treatment for necropsy. At postmortem examination, parts of neoplasms or organs with suspected neoplasm tissue were fixed for microscopic examination. Forty percent of the rats had tumors. The results were considered moderate in relation to the dose.

Montmorillonite

Heat-treated Montmorillonite in doses of 5, 15, and 45 mg was given to groups of 4 Sprague-Dawley rats by intratracheal instillation. ¹ Following a 3-mo postexposure period, the animals were killed and tissues were subjected to microscopic examination. The Montmorillonite particles were mainly restricted to alveoli within and adjacent to alveolar ducts regardless of dose. Most particles were contained within small to moderate numbers of pulmonary alveolar macrophages. However, some particles were free in alveoli. Adjacent alveoli septae were mildly thickened. Interstitial fibrosis was present in all groups. At the 5- and 15-mg doses, fibrosis was mild to moderate, multifocal, and loose, meaning less collagen. The 45-mg dose produced dense fibrosis. Macrophages contained clay particles and lymphocytes were present in the lesions. Occasionally giant multinucleate cells were seen.

Illite and Montmorillonite

The protective effect of Illite and Montmorillonite (up to 1 mg/mL each) on alterations in cell viability and epithelial barrier function induced by mycotoxins was evaluated using Caco-2 cells in a colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) assay. ⁵¹ Both clays provided protection against mycotoxin effects. Aflatoxin B1- and fumonisin B1-induced cytotoxicity were completely abolished by Illite. Decreases in the gene expression of specific claudin isoforms and the reduction of trans-epithelial electrical resistance of cell monolayers (an indicator of the epithelial barrier integrity) induced by mycotoxins were reversed by Illite. Montmorillonite also provided protection against mycotoxin effects, but at a lesser degree.

Illite, Montmorillonite, Kaolin

The cytotoxicity of an environmental dust sample (dust samples collected from a Mexican city) comprising approximately 75% Illite, Montmorillonite, and Kaolin, and approximately 20% α-quartz was studied in alveolar macrophages obtained from male albino Wistar rats. ³⁹ LDH release was used as an indicator of cell membrane disruption. The alveolar macrophages were incubated with and without the dust (3.2 µm mean diameter) for up to 2 h. LDH was measured in the supernatant at 0, 1, and 2 h, with controls run in parallel. Rat alveolar macrophages incubated with the dust released increasing amount of LDH into the medium as a function of time. Significant levels above control values (2.2 ± 2.6 LDH U/l) were observed by 1 h (19.5 ± 2.6 LDH U/l) and 2 h (29.5 ± 4.0 LDH U/li) of incubation.

The hemolytic activity of the dust was also investigated by incubating different particle concentrations (0.1, 0.5, 1.0, 1.5, and 2.0 mg/mL) with a 0.6% suspension of human red blood cells obtained from normal donors. Cell-particle suspensions were incubated at 37°C in PBS for 1 h under moderate agitation. The results indicated the dust induced significant hemolysis. An amount of 2 mg/mL produced 95 ± 3% hemolysis. The effect was observed starting at 1 mg/mL in a dose-related manner.

Kaolin

In a study examining the toxic mechanisms of typical fine particulate air pollution (PM_2.5), human bronchial epithelial (16HBE) cells were treated with nano-scale Kaolin at concentrations of 40 to 240 µg/mL. ⁵² The particle size information was not available; however, the authors stated the nano-scale Kaolin utilized in the study was to imitate Kaolin in atmospheric fine particles (PM_2.5). Cytotoxicity results of the cell counting kit-8 (CCK-8) assay showed the 16HBE cells had high viability after exposure to 40 µg/mL nano-sized Kaolin, but cell viability decreased significantly at doses greater than 80 µg/mL. A lactate dehydrogenase assay indicated that nano-sized Kaolin caused membrane disruption in a dose-dependent manner.

Bentonite

The ability for Bentonite (2/3 weight) and a zeolite (type not specified; 1/3 weight) to act as a hemostatic agent was studied in 12 male Sprague-Dawley rats. ⁵³ Another 12 rats served as controls. Approximately 8 g of the material was applied on wounded skin. Wounds were circular, full-thickness and 2 cm in diameter; skin samples were excised and evaluated stereologically after scarification. On days 12 and 21, 6 rats from the test group and 6 rats from the control group were killed. At day 12 termination, a reduction in the length density of the blood vessels (31%) and diameter of the large and small vessels (38% and 16%, respectively) was observed in the rats that received the test material. At day 21 termination, volume density of both the dermis and collagen bundles was reduced by 25% in the treated rats when compared to the controls. The researchers concluded the hemostatic agent containing Bentonite may cause vasoconstriction and inhibition of neoangiogenesis.

Attapulgite

In an intratracheal study, groups of 5 rats received a single instillation of Attapulgite at 1, 5, and 10 mg. ¹ One month after treatment, BAL and microscopic examination of the lungs were performed. The average length of the fibers was 0.8 µm, and 100% of the fibers were less than 3 µm. Every test animal had type A lesions. Type A lesions are characterized by an accumulation of inflammatory cells mostly macrophages, and epithelioid cells around fiber deposits. These inflammatory cells form a compact cellular infiltrate at the periphery of the deposits and some are focally dispersed throughout the alveolar region. The BAL had mostly macrophages and a small number of neutrophils at 5 and 10 mg doses. At the 5 mg dose, 3.6% of the cells were lymphocytes.

Groups of 5 male Wistar rats received 1, 5, or 10 mg of Attapulgite by transtracheal injection to examine alveolar macrophage production of interleukin-1 (IL-1) and macrophages-derived growth factor (MDGF) from fibroblasts. ¹ Saline and chrysotile B asbestos were used as controls. At 1 mo, Attapulgite produced granulomas and the chrysotile B produced fibrosis. At 8 mo, the granulomatous reactions had either resolved or were greatly diminished, whereas the fibrosis persisted. Cells obtained by BAL included multinucleated giant macrophages in animals treated with Attapulgite, but not in those treated with chrysotile B. Enhanced production of IL-1 was seen in all treated groups. MDGF production was only seen in animals with lung fibrosis.

Attapulgite with a mean fiber length of 0.8 µm and diameter of 0.02 µm was delivered to the lungs of sheep by bronchioscopic cannulation. ¹ The tracheal lobe of 16 sheep was subjected to a single exposure of 100 mg of Attapulgite in 100 mL of saline. A BAL was conducted at 2, 12, 24, 40, and 60 d, and necropsy was conducted on day 60. Total BAL cells, macrophages, and neutrophils, fibronectin content, and LDH and β-glucuronidase (β-GLUC) activity were examined. Nine samples of the tracheal lobe of the lung were obtained each time for microscopic examination. The controls were saline-exposed sheep and had no changes in BAL or pulmonary morphology. The total BAL cells/mL and subpopulations increased significantly above control numbers at days 12, 24, and 40 but returned to control levels by day 60. Albumin and procollagen III did not differ from controls, whereas fibronectin, LDH, and β-GLUC activities were significantly above the controls. Microscopic examination revealed infiltrates that were predominantly alveolar and peribronchial lesions. Macrophagic alveolitis with minimal airway distortion was seen. Three sheep had lesions of peribronchiolar alveolitis.

Bentonite

The ability of Bentonite to increase susceptibility to bacterial pneumonia was studied in mice. ¹ The animals were injected intratracheally with 1, 10, or 100 µg Bentonite. In vivo bacterial-infectivity screening assays were conducted by exposing the animals to aerosolized Group C Streptococcus species. The severity of infection was calculated by recording the deaths of the mice over a 15-d period. Control animals were exposed to titanium dioxide. At the 100 µg dose, Bentonite increased the infectivity of the bacteria. Mortality was 85%. Even at 10 µg, Bentonite caused increased animal mortality (43.3%). Control dust at 100 µg produced only a 5% mortality.

The effects of Bentonite dust in rats were analyzed in a 2-part intratracheal instillation study. ¹ A 0.5 mg dose of Bentonite with a mean particle size of 0.3 µm was instilled. Control animals were injected with sterile saline and titanium dioxide. Animals were killed at 1, 2, 6, 24, and 48 h and 4 and 7 d after instillation. Bronchopulmonary lavage was carried out and alveolar macrophages and polymorphonuclear leukocytes were recovered. The activity of LDH and protein content of the lavage fluid were also determined. In the first experiment, a rapid influx of polymorphonuclear (PMN) leukocytes was detected at 6 h. PMN leukocyte response peaked at approximately 19 × 10⁶ cells after instillation and started declining more slowly up to 4 d. At 7 d, the polymorphonuclear leukocyte numbers were 2.5 × 10⁶. The greatest increase in the numbers of alveolar macrophages recovered occurred at 4 and 7 d. The mean diameter of macrophages increased from 11.0 to 12.5 µm over the first 48 h after instillation. The mean diameter decreased at 4 and 7 d. LDH activity at 24 h was maintained at 40 milli units (mU)/mL and then increased (73 mU/mL) with the influx of polymorphonuclear leukocytes into the lungs after 48 h. Protein concentration was calculated at 500 µg/cm³ for the first 24 h and was maintained for 48 h.

In the second experiment, after instillation of 5 mg of Bentonite, the animals were killed at 1, 7, 49, and 100 d. ¹ In addition to the above measured parameters, peroxidase and lysozyme activity were also measured. A large number of polymorphonuclear leukocytes were recovered at day 1. However, the severity of the response did not differ significantly from the 0.5 mg dose. By 7 d, the numbers had decreased and were similar to control values. A significant decrease in the number of alveolar macrophages compared to controls was observed at 24 h after instillation. This decrease was followed by a sharp increase that exceeded control values by 7 d. Total number estimates were similar to those of the first experiment. LDH activity and protein concentration from Bentonite and titanium dioxide were very similar. The initial rise at day 1 following administration was short-lived. Peroxidase activity was minimal. Lysozyme activity rose sharply between 1 and 7 d, but returned to control values at 49 and 100 d.

In an intratracheal instillation study, a single dose of 40 mg of Bentonite suspended in 1 mL of physiological saline containing 40 000 IU of crystalline penicillin was administered to male CFY rats. ¹ The Bentonite composition consisted of 73% Montmorillonite, 18% cristobalite, 3% quartz, 3% feldspar, and 3% other minerals. Particle sizes were <2 µm. The control group received 1 mL of physiological saline containing 40 000 IU of crystalline penicillin. Animals were killed 12, 24, 48, or 72 h or 90 d after exposure. Body and lung weights of the rats were measured. The right lung was fixed and sectioned for microscopic examination. The lipids and phospholipids were analyzed in the left lung. The body weights of the rats were moderately decreased and the lung weight increased 72 h after Bentonite exposure. After 90 d, the lung weight was only slightly greater than that of the control animals. Upon microscopic examination at 12 h, Bentonite exposure had resulted in a nonspecific inflammation of mostly neutrophils with perivascular edema, alveolitis, and incipient bronchopneumonia. A small number of macrophages and lymphocytes were detected. Dust particles were observed in the leukocytes and macrophages or extracellularly in the alveoli. After 24 h, bronchopneumonia was present after coalescence of the inflammatory foci; the pneumonia then became necrotizing and desquamative. Necrotic neutrophilic leukocytes and eosinophil leukocytes were observed. The reticular network collapsed between 48 and 72 h. After 90 d of exposure, Bentonite caused storage focal tissue reaction (large foamy cells with pale cytoplasm). Closely-packed cells with dark cytoplasm and nuclei were located at the periphery. After 12 and 24 h, the amount of lipids and phospholipids in the lungs were not altered. However, between 48 and 72 h, the lipid and phospholipid content increased but distribution remained the same. After 90 d, the value was the same as seen at 72 h.

In another study by the same research group, male CFY rats were given a single intratracheal dose of 60 mg of Bentonite in 1 mL of physiological saline containing 40 000 IU crystalline penicillin. ¹ Bentonite particle size was less than 5 µm. Control groups received 1 mL physiological saline containing 40 000 IU penicillin. Animals were killed at the end of 72 h, weeks 2 and 4, and months 3, 6, and 12. The acid phosphatase activity and the progression of fibrosis were determined. The lungs were processed for microscopic examination and fibrosis determined by Belt and King’s classification. Acid phosphatase activity was increased at 72 h and had returned to normal by the first month. Loose reticulin fibrils, but no collagen, were observed after months 1–12.

Bentonite dust was administered intratracheally as a single 60-mg dose to Sprague-Dawley rats. ¹ The animals were killed 3, 6, and 12 mo after exposure. The right lung was studied microscopically and the lipids, phospholipids, and hydroxyproline values were determined. Significantly greater phospholipid values compared to controls were observed. Among the phospholipid fractions, the greatest quantitative increase was seen in phosphatidylcholine (more than twice the control) and the smallest increase was seen in phosphatidylethanolamine (less than 1.6 times). After 6 and 12 mo, the values were similar. Lung lipids had a greater range of values than did the phospholipids (no details given). The wet weight of the lung in grams increased in 5% to 10% Bentonite-treated rats compared to controls at month 3. No difference was detected at 6 and 12 mo. Hydroxyproline content of treated rats (mg/g lung wet weight) was very similar to controls at 3, 6, and 12 mo.

Subplantar injections of 0.05 mL of a 5% solution of Bentonite were given to male Wistar rats. ¹ The rats either received Bentonite injections in both hind paws at an interval of 24 h, or their left paw was injected with Bentonite and their right paw injected with 0.05 mL of a 10% solution of Kaolin (control). Subcutaneous Bentonite granulomas were produced on the left side, both dorsally and ventrally. Simultaneously, Kaolin granulomas were produced on the right side analogous to the Bentonite injection. Sodium salicylate and prednisone suppressed the Bentonite edema during the first 24 h. The presence of mononuclear cells was confirmed.

Kaolin

The ability of Kaolin to increase susceptibility to bacterial pneumonia was studied in mice. ¹ The animals were injected intratracheally with 100 µg Kaolin. In vivo bacterial infectivity screening assays were conducted by exposing the animals to aerosolized Group C Streptococcus species. The severity of infection was calculated by recording the deaths of the mice over a 15-d period. Control animals were exposed to titanium dioxide. A 100-µg dose of Kaolin caused statistically significant but modest (<50%) increased death due to infection by a large dose. Mortality was calculated at 38.9%. Control dusts at 100 µg produced only a 5% increase in mortality.

Clay

In an ocular irritation study performed in accordance with OECD TG 405, 100 mg of Clay (75% Illite, 16% Kaolin, and 9% Montmorillonite) was instilled into one eye of rabbits. ³⁸ The other eye served as a control and was instilled with 0.1 mL normal saline. No adverse effects were noted following treatment up to 72 h after instillation. The test material was considered to be non-irritating to rabbit eyes. No further details were provided.

Hectorite

In a primary eye irritation study using 9 New Zealand white rabbits, a 0.1 mL liquid or semisolid (100 mg of the solid) sample was instilled into the one eye of each rabbit. ¹ The eyes of 6 rabbits were not rinsed, and the eyes of 3 rabbits were rinsed approximately 4 s. All untreated eyes served as controls. The eyes were then examined with sodium fluorescein and an ultraviolet lamp at 24, 48, and 72 h and at 7 d. The mean score at 24 h was 2.0. All subsequent scores were 0.0. The test sample was considered moderately irritating to rabbit eyes without rinsing and practically nonirritating to the eyes with rinsing 4 s after instillation.

Kaolin

The potential for ocular irritation from a clay mask with 14.5% Kaolin was investigated in a tissue equivalent assay with EpiOcular™ cultures. ⁶⁵ The EpiOcular™ human cell constructs were exposed to 100 µl test material under standard culture conditions for 4, 8, 16, and 24 h. Tissue viabilities were then examined by MTT assay. The duration of exposure resulting in a 50% decrease of tissue viability (t₅₀) was calculated to be 5.2 h (at 4 h, tissue viability was 63.4%). The positive control yielded expected results. As residual test article may bind to the tissue and result in a false MTT reduction signal, a freeze-killed tissue control was used, and calculations were performed to correct for the amount of MTT reduced directly by the test article residues in the tissues. The clay mask with 14.5% Kaolin was predicted not to be an ocular irritant in this assay.

Montmorillonite

The effect of oral ingestion of Montmorillonite on protection against the adverse effects of the ingestion of aflatoxins were studied in 23 male and 27 female human subjects. ⁶⁶ The subjects received 1.5 g/d or 3.0 g/d in capsules. A total of 9 capsules were ingested over a 2-wk period. The study was randomized and double-blinded. Blood and urine samples were collected before and after the study. Mild gastrointestinal effects were reported with no statistical significance found between the treatment groups. No significant differences in hematology, liver and kidney function, or electrolytes were reported in either group.

Bentonite

Several case studies involving Bentonite workers have been reported. ¹ Some milling plants had dangerous concentrations of silica that ranged from 2 to 10 times the safe maximal concentration according to the US Bureau of Mines. Silicotuberculosis developed in four patients studied.

Fuller’s Earth

A patient that reported working no more than 15 yr in a Fuller’s Earth plant as a young man was diagnosed with terminal aspiration pneumonia, pneumoconiosis due to Fuller’s Earth exposure, bilateral emphysema, and fibrous pleural adhesions. ¹ The lesions differed from typical silicotic lesions of the lungs; no formations of the whorled, acellular collagen typical of silicotic nodules were observed. Isolated cavities in the apices were filled with black sludge and surrounded by vascular and cellular collagen. The dust in the lymph nodes had only stimulated the formation of reticulin fibers. No subpleural nodules were present. At mineralogical analysis, the Fuller’s Earth deposits were constituted mainly of Montmorillonite (85.2% to 90%).

Two additional cases of pneumoconiosis in employees that worked in processing or milling Fuller’s Earth for at least 28 yr were reported. ¹

Kaolin

A patient was reported with multiple pulmonary Kaolin granulomas. ¹ The man had a history of bilateral recurrent pneumothorax. Both pleural spaces were destroyed with a suspension of liquid Kaolin. Recurrent right-sided pneumothorax devolved and reobliteration was again performed. In a follow-up chest radiograph, multiple well-defined peripheral nodules were in both lungs and pathological analysis revealed a bland acellular material surrounded by chronic inflammatory cells. By light microscopy, the particles were consistent with Kaolin. It was presumed that Kaolin entered the lungs through pleuroalveolar or pleurobronchial openings.

In another investigation, the death of a 62-yr-old man who worked in a cotton textile mill for 43 yr was reported. ¹ The patient complained of progressive dyspnea and a productive cough. After being admitted to the hospital, a bronchoscopy was performed and no endobronchial lesions were found. A lung biopsy had lesions of severe interstitial fibrosis with bronchioalveolar structures extensively involved in the fibrotic process. Pathological alterations such as bronchiectasis, interstitial fibrosis with thickening of alveolar septa, mobilization of macrophages, and multinucleated giant cells were identified. Neither ferruginous bodies nor pleural hyaline plaques were identified. Kaolin particles were present with a mean size of 0.88 µm. Chrysotile asbestos was also detected, but the majority of particles were Kaolin. The man died as a consequence of respiratory failure despite an aggressive therapy of antibiotics and tuberculosis therapy.

The lungs and chest X-ray films were evaluated in a pair of case studies of men who worked in a Kaolin-processing plant for many years. ¹ The first case was a 36-yr-old man who worked on the plant for 17 yr. Chest films were taken at the end of his career and detected lesions of extensive confluent consolidation and nodule formation of advanced pneumoconiosis with infection. Autopsy and microscopic findings included alveolar spaces uniformly expanded, three areas of whorled fibrous tissue, scattered areas of cystic spaces, hilar nodes heavily pigmented, deposits of brownish black particulate matter, a large vessel with recent thrombus, hemorrhage, and necrosis, marked fibrous thickening of the pleura, and dense fibrous scarring of the lymph nodes. The final diagnosis was pneumoconiosis (kaolinosis) with pulmonary thrombosis and infarction of the lungs. The second case study was a 35-yr-old man who worked in the Kaolin-processing plant for 21 yr. Within his last 3 yr, he had dyspnea and a slight cough with small amounts of dark colored sputum. The sputum was negative for bacteria. Chest films revealed advanced pneumoconiosis with infection, confluent consolidation, nodular infiltration, cavitation, and emphysema. Autopsy and microscopic findings included nodules in the right and middle lobes, pleural spaces were thickened and shaggy, large bulbous emphysematous blebs, a pulmonary artery with organizing thrombus, heavily pigmented hilar lymph nodes, whorled fibrous collagenous tissue, and spaces and walls with macrophages. The final diagnosis was pneumoconiosis (kaolinosis).

A 35-yr-old man who worked at a Kaolin-processing plant for 17 yr presented with chest pain and was hospitalized. ¹ For the previous 2 yr before admittance, the man had packaged dried, processed Kaolin. Chest films revealed diffuse reticulonodular pulmonary infiltrates and a well-defined, noncalcified mass in the upper right lobe. A thoracotomy was performed and an 8 cm × 12 cm × 10 cm conglomerate pneumoconiotic lesion containing large amounts of Kaolin was found. X-ray diffraction material from the lesion had peaks corresponding to Kaolinite. The presence of silica was not confirmed by X-ray diffraction.

Pulmonary tissue was obtained from 5 Kaolin workers with advanced pneumoconiosis. ¹ Chest radiographs detected small irregular shadows and large opacities typical of Kaolin pneumoconiosis. At autopsy, firm, grey-brown nodules and masses were in the parenchyma and in the hilar lymph nodes. Microscopic lesions were extensive pulmonary Kaolinite deposition associated with the formation of peribronchiolar nodules. The nodules were composed of Kaolinite aggregates transversed by bands of fibrous tissue rather than dense whorled collagen. Kaolin was detected in the lungs. Silica was not detected by either analytical scanning electron microscopy or X-ray diffractometry.

Six additional cases of pneumoconiosis in employees of 12 yr in Kaolin processing or milling facilities were reported. ¹

Montmorillonite

A 73-yr-old Montmorillonite worker developed signs of pneumoconiosis, but subsequently died of acute gastrointestinal hemorrhage from a benign gastric ulcer. ¹ A chest radiograph taken 2 yr before his death showed a bilateral fine reticulonodular shadowing, while another radiograph taken a few weeks before his death indicated a slight increase in the reticulonodular opacities and a mass at the left hilum and apex. At autopsy, numerous soft stellate grey-black dust lesions 4–5 mm in diameter were observed occupying most of the lungs. No lesions of progressive massive fibrosis were identified. Also present were lesions of severe emphysema and a 4 cm diameter neoplasm arising from the bronchus of the left upper lobe. At microscopic examination, numerous interstitial collections of dust-laden macrophages were situated around the respiratory bronchioles and along the adjacent alveolar septa. A slight degree of fibrosis associated with the dust lesions was observed, and the neoplasm was a poorly differentiated adenocarcinoma containing giant cell areas. Mineralogical analysis showed a large amount of calcium Montmorillonite.

Bentonite

In a toxicological and occupational epidemiological review of Bentonite, the authors concluded Bentonite is probably not more toxic than any other inert insoluble dusts. ⁶⁹ However, because some forms may contain variable amounts of respirable crystalline silica, prudent management and adherence to occupational exposure limits is appropriate.