Animal Models

The rabbit ear is the most commonly used animal model. The rabbit's ear follicle has many structural similarities to the human sebaceous follicle. In 1941, Adams et al. [8] showed that the rabbit ear would respond similarly to human skin when exposed to chlorinated hydrocarbons, the most common cause of acne in industrial accidents. Until the rise of the animal rights movement in the late 1980s, cosmetic products were routinely screened using rabbits. Briefly, the method entails that the test product or ingredient is applied daily to the inner surface of one ear. This site is left open. The other ear is used as the negative control. At the end of either 2 or 4 weeks, the animal was sacrificed, and the degree of follicular hyperkeratinization is assessed. Frequently this was done by taking histological sections and giving an overall score based on the number and degrees of compacted follicles. Occasionally the impactions were removed from the ear using cyanoacrylate glue on a glass slide. This method is now frequently used in human testing.

The results from the rabbit-ear studies show that some cosmetic ingredients have comedogenic potential. These include branched chain esters and compounds that have solubility in both oil and water (hydrophile-lipophile balance (HLB) of 10-12) [9]. However, if these materials are chemically modified, or included at low levels with other ingredients in cosmetics, then their comedogenic potential is nullified. This was shown by Fulton [9] as well as Kligman and Mills [2]. Fulton showed that chemically modifying cosmetic ingredients can greatly effect their comedogenic potential. For instance, PEG-16 Lanolin gives a severe comedogenicity score of 4 on a 0 to 5 scale, whereas the higher-molecular weight and more water-soluble PEG-75 Lanolin yields a score of 0 under the

same test conditions (Table 1). Furthermore, Fulton showed that the comedogenic potential of fatty acid solutions was greatly reduced by replacing sunflower oil with acetone or ether as the solvent. Kligman and Mills reported that the comedogenic potential of vegetable oils is dose dependent, being abolished by diluting to 25% with mineral oil. From these observations, it appears vital to assess the comedogenic potential of the final product [7]. Additionally, Fulton and his colleagues screened the comedogenic potential of many ingredients and products [9,10]. Some of these results are included in Table 1.

It is interesting to note that the primary irritation potential does not correlate with comedogenic potential. For instance, sodium lauryl sulfate, which is frequently used as a model irritant, is noncomedogenic [11]. Conversely, many esters—such as isopropyl isostearate—that are highly comedogenic are relatively nonirritating.

To detect weak comedogens, the rabbit-ear assay was modified. Product applications were increased from 2 to 4 weeks, enabling the assay to detect products that cause comedones in a small but sensitive groups of consumers. The method may be overly sensitive for the average consumer, so there is a risk of false positives. Conversely, products that are noncomedogenic in the 4-week rabbit ear test are unlikely to cause comedone formation even in acne-prone consumers.

Many adverse reactions that consumers describe as breakouts or blemishes are not attributable to comedone formation. This is readily appreciated from the rapid onset of the blemish (a few days), which is too rapid for the formation of hyperkeratotic plugs in the follicular ducts. Furthermore, the formation of open or closed comedones frequently occurs without skin redness, whereas breakouts and blemishes described by consumers do have an inflammatory component. To better understand pustule formation, Wahlberg and Maibach developed a model to assess pustulogenic potential [12]. The test materials were placed on rabbits' backs and occluded for 24 hours. For some ingredients, the skin had to be abraded with a sterile needle to produce pustules. Pustule formation is dose dependent. Irritants such as sodium lauryl sulfate can elicit pustules even though they are noncomedogenic.

Human Models

Human models have been developed for looking at both acnegenic and comedogenic potential. Mills and Kligman first described the human comedogenic model in 1982 [13]. It is becoming more extensively used in the cosmetics industry as companies continue to avoid animal testing. In the human procedure, up to six test materials are applied to the upper back for 48 to 72 hours under an occlusive or, if necessary, a semiocclusive patch. Patches are applied three times a week for 4 weeks to give the 28 days of continuous exposure.

After induction, the test sites are sampled using an epidermal biopsy. A glass slide coated with cyanoacrylate (e.g., Crazy Glue®) is briefly applied to the skin for 1 minute. After it has dried the slide is removed, taking the follicular plugs and much of the stratum corneum with it. The size and number of follicular impactions are assessed using a 0-to-3 scale and compared with positive and negative controls. Positive controls include ace-tylated lanolin and coal tar. Mills and Kligman showed that the human model gave similar results to the 2-week rabbit-ear method (Pearson r = 0.944, n = 32 cosmetic ingredients or products). However, the rabbit-ear model appears to be somewhat more sensitive than the human assay (Table 2).

Recently, a new method for epidermal biopsies has been validated. The method uses

Table 1 Comedogenicity and Irritation Potential of Cosmetic Ingredients in the Rabbit-Ear Model

Ingredient

Comedogenicity

Irritation

Oils

Cocoa butter

4

0

Coconut butter

4

0

Evening primrose oil

3

2

Soyabean oil

3

0

Peanut oil

2

0

Castor oil

1

0

Sunflower oil

0

0

Mineral oil

0-2

0

Lanolin and derivatives

Acetylated lanolin

0

0

Acetylated lanolin alcohol

4

2

Anhydrous lanolin

0-1

0

Lanolin alcohol

0-2

0

PEG-16 Lanolin

4

3

PEG-75 Lanolin

0

0

Fatty acids and esters

Lauric acid

4

1

Myristic acid

3

0

Palmatic acid

2

0

Stearic acid

2-3

0

Butyl stearate

3

0

Cetyl acetate

4

2

Cetyl ester NF

1

1

Isopropyl isostearte

5

0

Isopropyl lineolate

4

2

Isopropyl myristate

5

3

Alcohols, sugars, and their derivatives

Isopropyl alcohol

0

0

Cetyl alcohol

2

2

Isocetyl alcohol

4

4

Oleyl alcohol

4

2

Stearyl alcohol

2

2

Sorbitol

0

0

Sorbitan laurate

1-2

1-2

Sorbitan oelate

3

0

Sorbitan stearate

0

0

Oleth-3

5

2

Oleth-5

3

2

0leth-10

2

1

0leth-20

1

0

Source: Ref. 9.

Table 2 Comparison of Human-Back and Rabbit-Ear Comedogenicity Scores

Mean comedogenicity score

Material

Rabbit*

Human

Acetylated lanolin alcohol

3

2

Cocoa butter

3

2

5% crude coal tar**

3

3

Isopropyl myristate

1

0.4

Safflower oil

1

0

5 or 8% sulfur**

3

2

2.5% sulfur**

2

1.2

Hydrophilic ointment

0

0

* Comedogenicity scored on a 0-3 scale, n = 3 rabbits and 5 humans.

** These test material were diluted with hydrophilic ointment. All other test materials used at full strength. Source: Ref. 13.

* Comedogenicity scored on a 0-3 scale, n = 3 rabbits and 5 humans.

** These test material were diluted with hydrophilic ointment. All other test materials used at full strength. Source: Ref. 13.

commercially available cosmetic strips that are designed to remove impactions from the face without damaging the skin [14]. The biore pore strip®, which uses a cationic polymer, preferentially interacts with the proteins of the hyperkeratotic plugs but not the stratum corneum. The plugs have more acidic amino acids and are therefore more negatively charged than the surrounding stratum corneum. An example of the biore pore strip removing impactions from the nose is shown in Figure 2. Rizer et al. showed that the biore pore strip® removed over 70% of the impactions that cyanoacrylate removes, but without the damage of the latter. The biore pore strip is more effective than other cosmetic strips in removing plugs from the follicles. The other strips use nonionic polymers, which are not able to preferentially interact with the follicular plugs.

Figure 2 Half a biore pore strip under UV light. The hyperkeratotic impactions on the strip fluoresce due to the P. acnes.

One advantage that the biore strips have over the cyanoacrylate glue is that it is far less damaging and irritating to the skin. Therefore, it can be used to measure comedone formation on the face at the end of usage studies. Cyanoacrylate is too damaging to be acceptable to most test panelists for use on their face. The biore pore strip has been shown to be acceptable to panelists; indeed, this is its intended use.

Human Usage Tests

Ultimately, all predictive models must be related to back to the consumers' experience in the marketplace. Consumers who experience an adverse reaction will report it in terms most familiar to them. Most consumers do not differentiate between acne and comedone formation, or blemishes and breakouts.

One approach to assessing the rate of adverse reactions is to have a group of consumers use a product for several weeks [15-18]. Test subjects should be evaluated for comedones, pustules and papules at the beginning of the study, and then at set intervals. A 1-week evaluation will reveal any propensity to cause irritation, including follicular irritation that panelists may recognize as breakouts. Any sensory irritation will become evident during the first week. Three and six-week evaluations are used to detect comedogenicity and acnegenicity. This design is consistent with the recommendation of the American Academy of Dermatology's consensus panel on acnegenicity testing [19].

A sizable proportion of adverse reactions is experienced by vulnerable subgroups. A subgroup for irritation may include panelists whose skin is readily irritated by surfactants. Another subgroup will include panelists with acne-prone skin. Both subgroups should be identified and form a significant part of the test panel. This will enable the investigator to identify potential problems before the product reaches the marketplace.

SUMMARY

The induction of comedones and acneform eruptions is a significant concern to many consumers, especially those with acne-prone skin. Any product that has a propensity to produce these eruptions will be unsuccessful in the marketplace. Indeed, many consumers expressly look for products that are labeled noncomedogenic and/or nonacnegenic.

Cosmetics manufacturers are meeting this consumer demand by showing that their products do not cause comedones and/or acne breakouts, and label their products accordingly. Consumers judge a facial cosmetic on whether it causes breakouts, blemishes, bumps, or blackheads. There are multiple causes for the reaction, including comedone formation and follicular irritation. Consumers do not differentiate between the biological mechanisms; they are only concerned with the results they produce.

Today, human models have replaced animals for testing both comedogenicity and acnegenicity. Comedone formation is determined by continuously patching the material on the human back for 28 consecutive days. Comedones are quantified by extracting the plugs from the follicle using cyanoacrylate glue on a glass slide or a biore pore strip® containing cationic polymers. The degree of the impactions is compared with positive and negative controls.

Acnegenicity is assessed by human-use testing, where a panel of consumers uses the product under normal conditions. The skin is evaluated by a trained observer for comedones, papules, and pustules at the beginning of the study, and then after 1, 3, and 6 weeks of usage. Cosmetic pore strips such as biore® can be used to assess comedone formation.

These can remove follicular plugs from the face without the skin damage associated with cyanoacrylate.

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