Sociedade Brasileira de Dermatolodia Surgical & Cosmetic Dermatology


ISSN-e 1984-8773

Volume 1 Number 4

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Continuing medical education

Hair care: a medical overview (part 2)

Tratamentos estéticos e cuidados dos cabelos: uma visão médica (parte 2)

Leonardo Spagnol Abraham1, Andreia Mateus Moreira1, Larissa Hanauer de Moura1, Maria Fernanda Reis Gavazzoni1, Flávia Alvim Sant’Anna Addor1

Dermatologist – Brazilian Society of
Dermatology1, MD in Dermatology – Universidade
Federal do Rio de Janeiro2, Dermatologist – Brazilian Society of
Dermatology1, PhD and MD in Dermatology –
Universidade Federal do Rio de
Janeiro4, 5MD in Dermatology – FMUSP;
Associated Professor at Dermatology
Department – UNISA5



The authors complement the fi rst article on the approach of dermatological aesthetic hair treatments. This article addresses the ultimate hair straightening with major straightening legalized – sodium, lithium and guanidine hydroxides and ammonium thioglycolate –, their differences, mechanisms of action, indications and safety to human health. Besides these straighteners, we discuss the illegal and indiscriminate use of hairdressers salons in Brazil in the group of aldehydes (formaldehyde and glutaraldehyde) for smoothing hair. This study focuses its legal implications and public health, carcinogenic potencial and the identification of such filings with ANVISA. Also studied the shampoos and conditioning agents indicated for treatment of the hair shaft. Finally, we discuss the implications of hair care in general health of the hair shaft and the scalp.

Permanet hair and straightening


Hair straightening is a temporary or permanent breaking of chemical bonds that keep the tri-dimensional structure of keratin molecule in its original rigid form. These are divided in strong (disulfi de bridges) and weak bonds (hydrogen bridges, ionic bonds and van der Waals forces). Weak forces are broken in the simple act of wetting the hair. The weaker chemical bonds result from attraction between positive and negative charges. There are temporary hair straightenings, which use physicochemical techniques as the hair drier and piastra (“fl at iron”) and also the technique of hot comb. They are temporary because they last until the next hair washing. Hair need to be previously wet, in order to break hydrogen bridges in the process of keratin hydrolysis, allowing the temporary opening of these bridges helicoidal structure. With this process hair becomes straight. The rapid dehydration of hair dryer maintains the hair straight shape. Application of fl at iron shapes cuticle cells (scales) as if fl attening them parallel to the hair stem. The hair gets straight and shiny as it refl ects more incident light.1

Permanent hair straightening aim to break the disulfi de bridges of keratin. They may be based on sodium hydroxide, lithium and potassium hydroxide, guanidine (calcium hydroxide + guanidine carbonate), ammonium bisulfi te and thioglycollate, or ethanolamine, which use chemical reactions of reduction.2,3


Sodium hydroxide, lithium hydroxide, and guanidine hydroxide (composed of guanidine carbonate and calcium hydroxide) are the most powerful and are intended, in general, to Afro-ethnic hair. The fi rst is used in concentrations ranging from 5% to 10%, at pH 10% to 14%, promoting the most dramatic results and is the one causing more hair damage. Guanidine hydroxide is less potent than sodium hydroxide, but still has high potential to damage the hair fi ber. They act by promoting the breaking of keratin disulfi de bridges in a process called lanthionization, which is the replacement of 1/3 of amino acid cysteine by lanthionine. Hair is composed of approximately 15% of cystine.4 Alkaline pH (between 9 and 14) is used, which causes the swelling of fi ber, allowing the opening of the outer layer, the cuticle, so that relaxer penetrates this and the next layer, the cortex. After that, a substance that acidifies pH is applied interrupting the process and closing again the disulfide bridges in the desired hair new format. Generally, acid shampoos are used for this purpose (pH between 4.5 and 6.0).


Ammonium or ethanolamine thioglycollate belongs to the “thiols” family and is the most used in Brazil. It is far less potent than sodium hydroxide and, in general, milder than guanidine. It has the highest cost among all relaxers. Its concentration depends on the pH of ammonia solution. If a thioglycollate solution 6% at pH 9.8 is used, the same action power of a 10% solution at pH 9.35 is achieved, but the first solution is potentially more abrading, and due to the higher concentration of ammonia, it has a much more unpleasant odor. In most cases, a solution between 7.5% and 11% at pH between 9 and 9,3 is used. It can be applied to dry (preferably) or wet hair. The product concentration should be chosen according to hair type (Table 1).4

Thioglycollate is currently the most used compound for Caucasian hair straightening. It breaks the disulfide bridges of cysteine amino acids, causing the formation of two cysteines for each cystine. Through this process, keratin suffers edema and becomes malleable enough to be curled (perm) or straightened. In perm process, waving rollers or rods are used, and in hair straightening a hairdryer is used first and then a flat iron is applied to stretch the hair. A better straightening is achieved by applying the flat iron in very thin locks of hair. Afterwards, the hair is washed with tap water and thioglycollate is neutralized with the application of an oxidative agent, usually containing hydrogen peroxide. The chemical process is then stopped, with hair strands being kept permanently in the new format. The entire process can last up to 7 hours, if flat iron was applied in very thin locks of hair. The so-called “relaxation” is the application of thioglycollate without the use of flat iron. It is a faster process, but the smooth effect is less dramatic. In chemically treated hair, an application of the thioglycollate should be done in a strand test before starting the process to determine the hair resistance to product. Application of permanent coloring or toning can be done about 15 days after straightening. It should be noted that relaxed hair become more susceptible to chemicals, especially to bleaching. Thioglycollate is not compatible with hydroxides, and simultaneous application of both products on the same area will result in the hair tonsure.5,6

Light Emitting Diode (LED)

LED is a semiconductor diode (P-N junction) that emits visible light when energized. The light is monochromatic and produced by electron energetic interactions. The light emission process through the application of an electric source of energy is called electroluminescence. In any P-N junction directly polarized within the structure, near the junction, there is gaps recombination of electrons. This recombination requires the energy possessed by the electron, which until then was free, to be liberated, which occurs as heat or light photons. There are techniques using thioglycollate associated with LED application in hair already submitted the straightening. The proposal would be that the light would help the penetration of thioglycollate and also, by itself, generate breaks in disulfide bonds of keratin, which would allow the use of lower concentrations of straightening with less damage to the hair and more power of straightening. In medical literature there are no studies to support this result, wich would scientifically justify the use of LED in association with thioglycollate.


Use of formalin for hair straightening has become frequent because, besides being cheaper, it is a quick process and leaves hair with an intense glare. Indeed, formalin is a formaldehyde solution at 37% and its sale in pharmacies is prohibited. The solution is empirically mixed with liquid keratin, which consists of positively charged amino acids, and cream conditioner. The final product is applied on hair strands and spread with the help of a comb. Then a hairdryer and flat iron are used. Formaldehyde binds to cuticle proteins and amino acids hydrolyzed keratin solution forming a hardener film along he hair, waterproofing and keeping it hard and straight (Figure 1). The effect is the same as in caramel apple: beautiful and bright outside, but dehydrated and brittle inside (Figure 2). The hair is susceptible to fracture as a result of daily normal trauma, such as combing, brushing, and tight the hair. The biggest problem is that formaldehyde is volatile and, after heating, a larger amount is inhaled by both those who apply and those undergoing treatment. Formaldehyde is allowed in cosmetics in concentrations up to 0.2% as a preservative and 5% as a nail hardener (ANVISA - Legislation in force: Formaldehyde preservative, RDC Resolution No. 162 of 11 September 2001 and formaldehyde as nail hardener: RDC Resolution No. 215 of 25 July 2005), but its use as a hair straightening is not allowed due to volatilization. Recently, a new resolution was issued prohibiting its use for this purpose (ANVISA - RDC Resolution No. 36, June 17, 2009). To achieve the smoothing effect, formaldehyde should be used in concentrations of 20% to 30%, which is totally banned.

Glutaraldehyde is a colorless liquid slightly acidic in its natural state, which is being commonly used as a hair straightener since the ban of formaldehyde. It is a clear liquid found in aqueous solution at 50%. After activation with baking soda to make the solution alkaline, the liquid becomes green. In Brazil, after dilution, it is marketed as sterilant and disinfectant for hospital use at concentrations of 2%. Glutaraldehyde (glutaral) is a relatively common preservative in cosmetics, and can be used in concentrations up to 0.2%. Its activity is due to the alkylation of sulfhydryl, hydroxyl, carboxyl, and amino groups, altering DNA, RNA, and protein synthesis. Mutagenicity of glutaraldehyde is very similar to that of formaldehyde. The inhalation exposure to glutaraldehyde and formaldehyde results in damage to upper respiratory tract epithelium. Glutaraldehyde is 6 to 8 times stronger than formaldehyde to produce protein cross-links in DNA and about 10 times stronger than formaldehyde to produce tissue damage inside the nose after inhalation. The International Agency for Research on Cancer (IARC) classifies the substance in group 2A; that is, as a probable human carcinogen. Yet, the New Zealand Nurses Organization believes that glutaraldehyde is a neurotoxic, leading to memory loss and concentration difficulty, in addition to tiredness and fatigue.6

Formaldehyde Risks

The risk of formalin in its inappropriate application is proportional to its concentration and frequency of use (the higher the concentration the bigger the risk). Inhalation of gases and contact with skin is dangerous for both professionals and users (Table 2).

Inhalation of this compound may cause irritation to eyes, nose, mucous membranes and upper respiratory tract. In high concentrations may cause bronchitis, pneumonia, or laryngitis. The most frequent symptoms in case of inhalation are severe headaches, coughing, shortness of breath, dizziness, difficulty breathing, and pulmonary edema. Contact with vapor or solution can leave the skin whitish, rough, and cause strong sense of anesthesia and superficial skin necrosis.

Long periods of exposure can cause dermatitis and hypersensitivity, skin rashes (dryness) and ulcerations, mainly between the fingers. It can also cause conjunctivitis. The formaldehyde vapor irritates all parts of the upper respiratory system and also affects the eyes. Most people can detect formaldehyde at concentrations as low as 0.5 ppm, and, as the concentration is increased until the current Maximum Exposure Limit, the irritation is more pronounced (Table 3).7

Formaldehyde concentration measurements in the air of pathology laboratories have shown levels between 0.07 and 2.94 ppm (parts per million). Environments in which the substance is used can not have more than 0.019 mg/m3 in the air and, certainly, the levels exceed this limit after heating.

Carcinogenicity, the assessment of carcinogenic potential, of formaldehyde was assessed by 4 international research institutions and was classified in 1995 by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1, July 2004), tumorigenic, and teratogenic because it produces reproductive effects in humans. The USA Environmental Protection Agency (EPA) and the Occupational Safety and Health Association (OSHA) believe that the agent is suspected of causing cancer in humans. The USA National Toxicology Program (Fourth Annual Report on Carcinogens) reported in 1984 that formaldehyde is a carcinogenic agent to rats in the following doses: oral, 1170 mg/kg; dermal, 350 mg/ kg; and inhalation, 15 ppm/6 hours.8

Hair straightening products are registered as cosmetic with degree of risk 2 by the Brazilian drug administration organ (ANVISA), i.e. they require registration for marketing. However, a practice now considered illegal and prohibited is the addition of formaldehyde or glutaraldehyde in these products to increase straightening capacity. To determine whether a product is registered as a degree 2 cosmetic, simply visit the site of ANVISA, using the following path: http:// cosmetico.asp. Through the trade name or registration number appearing on the label, it is possible to know if the product is illegal or not. This number starts with the numeral 2, and has 9 or 13 digits (Figure 3).9

Final Consideration on Hair Straighteners

Once hair is straightened by the application of guanidine or sodium hydroxide it can not be straightened again because there is the risk of breakage. Now when thioglycollate is used, it is recommended to straighten only the newly grown hair, however, when using a low concentration, a new application is required and a careful strand test should be done before the whole process. Straightened hair can only be subjected to another straightening process with the same substance used initially. Hair straightening should be done at least 2 to 4 weeks prior to hair coloring.

The hair straightening process know in Brazil as “progressive brush” (without formalin) is the application of thioglycollate every 4 months or so, to a progressive straightening effect. While “Japanese brush” is the application of thioglycollate in high concentration for a fast straightening in only one session. Hair straighteners should not be applied directly to the scalp, and for the most potent as sodium hydroxide, the skin should be protected with prior application of oil or petroleum jelly.

Hair Shaft Care


Currently, the goal of shampoo is not only to remove sebum, sweat, debris, ions, hair products fatty acids, oxidized metal particles, and impurities from the scalp, but also to help hair aesthetics. In our days a shampoo can have more than 30 ingredients in its formula, since, there are the conditioning agents to minimize hair aggression, besides the surfactants, which are cleaner agents. These agents can be lipids, carboxylic acids, cationic agents and silicones (dimethicone, amodimethicone).1

The main element of a shampoo is a surfactant or detergent, which is a molecule with a non-polar or hydrophobic portion, which binds to lipids of sebum and other impurities, and a polar or hydrophilic portion, which connects to the water allowing the removal and rinse of desired material. There are four basic categories of surfactants: anionic, cationic, nonionic, and amphoteric (Table 4). Each of these groups has different qualities of cleaning the scalp and conditioning the hair. Typically, many surfactants are combined into a single formula to achieve the desired result.10

Anionic surfactants such as sodium lauryl sulfate and ammonium laureate, ammonium sulfate and alpha-olefin sulfonate are the most widely used commercially. They are excellent for removing sebum from the scalp. However, they are not well accepted by consumers because of its unsightly result to hair, living it opaque, slightly malleable, and difficult to comb. To keep the power of a cleanser shampoo, the withdrawal of hair natural sebum is minimized, and many formulas add other secondary surfactants as the nonionics.

Cationic surfactants are used in shampoos for dry or chemically treated hair due to their limited power to remove sebum, and for keeping the hair soft and malleable. The most common cationic surfactant is cetyl trimethyl ammonium chloride. It forms positively charged ions when in aqueous solution and has cleansing properties and weaker foaming power than anionic. For this reason, a coconut type chain can be added to cationic surfactant to allow greater amount of foam.

Non-ionic detergents are used in combination with anionic as secondary cleaners, as they present a small capacity to clean the scalp. Their goal is to soften the anionic surfactant. Some examples are PEG-80 sorbitan laurate and cocoanphocarboxyglycerinate.

The last surfactant category consists of amphoteric detergents, which are substances that present both negative and positive poles. This causes them to behave as cationic detergent at low pH and as anionic at high pH values. Examples are: cocodietanolamin, betaines, cocoamidopropylbetaine and cocoamphoacetate. This type of surfactant is used in shampoos for babies, since it does not irritate the eyes and is also suitable for thin hair.11

Currently, there is a strong negative advertisement about the presence of salts in shampoo formulas. Salts such as sodium chloride or similar is needed to control viscosity. If salts are not present in quantities above the ideal, they do not offer risk to hair health. In very high quantities they are substances that, by having a positive charge, compete with the polymer for the same binding site, thereby reducing the power of conditioning products. However, even in high amounts they offer the benefit of removing polymer residues deposited between scales.

Shampoos and chemical treatments increase the hair electrostatic charge (negative charge), which is compensated by applying a cationic conditioner (positive charge). This leads to accumulation of waste in cuticle, particularly endocuticle, in junction areas between the cell layers, called cell membrane complex. Such waste increases hair static electricity, raising the scales, leaving the hair frizzy, difficult to comb, and unsightly. In addition, the fatty acids in conditioning shampoos and conditioners bind to calcium and magnesium present in shower water and are deposited on hair fiber. Therefore, the weekly application of anti-residue shampoo is needed; whose high power astringent surfactant is ammonium lauryl sulfate.


They are substances designed to detangle hair, facilitate hairstyle, and reduce aggression of physical and chemical effects to which hairs are subjected daily, such as the simple act of combing, keeping the aesthetic aspect of hair, the softness, and reducing the appearance of rebellious hair (frizz effect). Conditioners consist of mineral and vegetable oils, waxes, long chain alcohol, cationic substances (positively charged), triglycerides, esters, silicones and fatty acids.12

The intention is to bind these substances in damaged areas of cuticle and cortex. Binding and interactions of cosmetic components with keratin are influenced by the electrical charge of each ingredient, molecular size, isoelectric point of hair fiber, and ingredients previously adhered to cuticle. The common ingredients to conditioners are stearalkonium chloride, sodium chloride cetrimonium, dicetyldimonium chloride, Polymer JR (polyquaternium-10), quaternary polymers, cationic guar gum, acrylamide, methacrylate, neutral polymers (copolymers, polyvinyl), and fatty acids (lauric, myristic, palmitic ).

The ingredients can act both on the hair surface and deeply in the cortex. Substances that act in the cuticle have a high potential for adsorption, which means a form of adherence or coating, also called a film effect. Substances of high molecular weight act more in the cuticle, while the low molecular weight substances penetrate the hair inner layer.

There is also influence of pH, since acidic substances act more quickly, with detangling effect by decreasing the electrostatic charge. Alkali substances penetrate deeper in the cortex and act more slowly. The former are conditioners and the others are fast action products (1 to 3 minutes), and the others are the masks that require at least 15 minutes of application. Ideal conditioner should have pH between 4.1 and 4.9. Unfortunately, it is not required to specify the product pH in packaging.

One widespread category among conditioning agents are polymers, which are substances found in shampoos, conditioners, creams with and without rinsing, mousses and fluids. Their function is to reduce the hair electrostatic charge and increase the hair strand substantivity forming a protective film.

Polymers bind to hair in three ways (ionic and covalent, hydrogen bonds, and attractive forces of Van der Waals) and are classified as:

a) cationic: polyquaternium ammonium, dimethyl ammonium, stearalkonium chloride, or cetrimonium chloride;

b) mono and polypeptides: hydrolyzed proteins (amino acids), polypeptides derived from collagen.

The molecular weight and size of polymer influence the absorption and dispersion through the hair fiber and its binding to keratin. Polymers of low molecular weight diffuse themselves inside the fiber (MW 10,000 to 250,000), and polymers of high molecular weight diffuse themselves on the fiber surface (over 250,000).

Moreover, the hair electric charge allows a greater or lesser binding of polymers. Most hair cosmetics have pH more alkaline than the hair strand pH, charging them negatively and facilitating the binding of cationic polymers. Cationic polymers are difficult to remove by ordinary shampoos (residues). The polypeptides bind to the fiber because they have many ionic points and sites of polar bonds. They are large molecules and have binding areas for Van Der Waals forces; while monomers of proteins (methionine, tyrosine, tryptophan) when in aqueous solutions have low molecular weight and penetrate inside the fiber.

Currently, the most widely used agent and conditioner is silicon. Silicones as cyclopentasiloxane, dimethiconol, dimethicone, and amodimethicone have a film effect and protect the hair from high temperatures of hairdryer and flat iron, as the heat spread along the fiber. They also reflect light, which increases the shiny. Silicones help to flatten the cuticle anucleate keratinocytes, causing the scales not detached from each other, helping to keep the hair untangled.

Recently, hair care treatments included the use of sunscreens. They were first used for preserving hair products from the action of sunlight, but today it is known that these filters act on the hair strand while minimizing degradation of tryptophan and the breaking of disulfide bridges by UVB rays through absorption. Examples are incroquat UV 283, merquat, and escalol HP 610. Sunscreens such as quaternized chloride cinamidepropyl trimethyl ammonium and dimethyl- PABA-amidopropyl-laurdimonium tosylate have a positive charge that binds to hair strand which is negatively charged, forming a protective film coverage. All these products should be reapplied when the hair is wet.

It is noteworthy that topical application of nutrients, vitamins, pro-vitamins, panthenol, among others substances announced in shampoos and conditioners, do not alter the hair strand structure, and there is no scientific evidence of benefit in their use.

Implications of Cosmetic Treatments on Hair Shaft

The medical literature reports some possible effects after hair straightening: shaft fracture (usually at the point of junction of pre-treated hair with the new hair being chemically treated, or even in distal part of the hair) (Figure 4), scarring alopecia, follicular degeneration syndrome (hot comb alopecia, whose name tends to be replaced), induction of telogen effluvium, and possible damage to hair follicles.13,14

Moreover, there are frequent scalp burns occurring when the product is applied directly to skin. The permanent dyes using paradiamine oxidation are the main causes of contact eczema. The most involved in allergy are: p-phenylenediamine, p-chloro, and toluenediamine phenylenediamine. Formaldehyde can also cause contact eczema. Eczema condition starts in scalp periphery and behind the ears, followed by scalp itching. Lesions may extend to face, particularly in eyelids and periocular region.

P-phenylenediamine can induce asthma in hairdressers. There is a concern about the systematic use of hair coloring products. Studies to investigate the carcinogenic potential of various types of dyes did not consider the substances currently available in the market as being risky. The substance 2,4-diamino anisole was withdrawn from the market due to its relationship to carcinogenicity. New studies on the toxicological potential of hair dyes are still in development, covering larger populations and more prolonged use of dyes.

Chemicals used for hair straightening, permanent, or coloring leave hair rough, porous, and brittle, with lower tensile strength due to the generation of negatively charged ions along the keratin molecule. Shampoo also removes excess sebum and natural fat present along the hair strand.

Small everyday traumas, to which hair is subject such as the act of combing and brushing, also generate the production of negative ions in cuticle and cortex, particularly in chemically treated hair.15-18

To minimize these effects, conditioning agents are used after washing to keep hair soft, easy to comb, shiny and silky. Conditioners reduce static electricity and friction between the hair strands detangling them because they deposit ions positively charged inside the hair, which bind to negatively charged ions and neutralize them. Friction is then reduced, causing an increase in adherence of cuticle scales and, therefore, the hair is more reflective of incident light and silky in touch. There are several types of conditioners: instant, deep (to rinse), and “leave-in” (without rinsing); the components of the first two should be resistant to subsequent rinse.19

During pregnancy and lactation, it is not recommended the use of any type of chemical for hair coloring, perming, or straightening, even with henna. There is no consensus about the fetus safety during the use of such techniques and substances, but it is known that the risk is higher for professionals who apply chemicals without wearing gloves and masks that give adequate protection. There are reports on cases of neuroblastoma and congenital cardiovascular diseases caused by mothers exposed to dyes, permanent and toning. Some authors consider henna to be mutagenic and capable of living residues on scalp.20


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2 . Robbins CR. Chemical and physical behavior of human hair. Springer. 4 ed. 2002.

3 . de Sá Dias TC, Baby AR, Kanko TM et al. Relaxing/straightenig of Afro-ethinic hair: historical overview. J Cosmet Dermatol 2007; 6(1):2-5.

4 . Bouillon C, Wilkinson J. The science of hair care. Taylor & Francis. 2 ed. 2005.

5 . Cannell DW. Permanent waving and hair straightening. Clin Dermatol 1988; 6(3): 71-82.

6 . Wickett RR Permanent waving and straightening of hair. Cutis 1987; 39(6):496-497.

7 . Lewis RJ, Tatken RL. Registry of Toxic Effects of Chemical Substances. On-line Ed. National Institute for Occupational Safety and Health. Cincinnati, OH. March 13, 1989.

8 . (acessado em 26/10/2009)

9 . (acessado em 26/10/2009)

10 . Bolduc C, Shapiro J. Hair care products: waving, straightening, conditioning, and coloring. Clin Dermatol 2001; 19(4):431-436.

11 . Nagahara Y et al. Structure and performance of cationic assembly dispersed in amphoteric surfactants solution as a xampu for hair damaged by coloring. J Oleo Sci 2007; 56(6):289-295.

12 . Bouillon C. Shampoos and hair conditioners. Clin Dermatol 1988;6:83-92.

13 . Dawber R. Hair: its structure and response to cosmetic preparations. Clin Dermatol 1996; 14(1):105-112.

14 . Nicholson AG, Harland CC, Bull RH, Mortimer PS, Cook MG. Chemically induced cosmetic alopecia. Br J Dermatol 1993; 128(5):537-541.

15 . Nicholson AG, Harland CC, Bull RH, Mortimer PS, Cook MG. Chemically induced cosmetic alopecia. Br J Dermatol 1993; 128(5):537-541.

16 . Callender VD, McMichael AJ, Cohen GF. Medical and surgical therapies for alopecias in black women. Dermatol Ther 2004; 17(2):164-176.

17 . Trüeb RM. Hair loss. Praxis (Bern 1994) 2003; 92(36):1488-1496.

18 . Scott DA. Disorders of the hair and scalp in blacks. Dermatol Clin 1988; 6(3):387-395.

19 . Gummer CL. Cosmetics and hair loss. Clin Exp Dermatol 2002; 27(5):418- 421.

20 . Blackmore-Prince C, Harlow SD, Gargiullo P, Lee MA, Savitz DA. Chemical hair treatments and adverse pregnancy outcome among Black women in central North Carolina. Am J Epidemiol 1999; 149(8):712-716.

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