The use of the challenge test to analyse preservative efficiency in non-sterile cosmetic and health products: applications and critical points

Abstract

Here we review the challenge test, which is used to evaluate the efficiency of preservatives in non-sterile cosmetic products. We describe the historical context, explain the technique and interpretation of the results according to pharmacopoeias and emphasise the critical points of the technique. In order for microbial growth to occur, non-sterile formulations must have water, minerals and vitamins in addition to other nutrients. The challenge test is a useful guide regarding the type and amount of preservatives to be added to a product to ensure its quality. This technique can also be used to analyse the efficiency of the product packaging and whether it prevents microbial contamination. This method is used in security and stability tests during product development, but it is not routinely performed as a control. Although there is no consensus regarding its use, this test is the most suitable to analyse preservative effectiveness.

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Introduction

Thousands of people are affected each year worldwide as a consequence of non-sterile cosmetic products, making the development of effective analyses and quality control measures imperative.^1,2

According to the Food and Drug Administration (FDA), non-sterile products can be classified as either Type I or Type II according to their application. Although the products may contain microorganisms, these guidelines provide maximum allowable limits and require the absence of pathogens such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Salmonella sp. and Aspergillus sp. to maintain product quality and avoid risk to the consumer.³

According to the FDA, products used in the eye area, in sensitive areas such as the mucosa and in infants are classified as Type I. The acceptable limits for microorganisms in Type I products are: (1) total aerobic mesophiles must not exceed 5 × 10² colony forming units (CFU)/g or /mL; (2) there must be a complete absence of Pseudomonas aeruginosa, Staphylococcus aureus and faecal coliforms in 1 g or 1 mL; and (3) Clostridiumsulfite-reducers (exclusively for talc) must be absent in 1 g.³

For Type II non-sterile products, the acceptable limits of contamination are: (1) total aerobic mesophiles must not exceed 5 × 10³ CFU/g or /mL; (2) there must be a complete absence of Pseudomonas aeruginosa, Staphylococcus aureus and faecal coliforms in 1 g or 1 mL; and (3) Clostridiumsulfite-reducers (exclusively for talc) must be absent in 1 g.³

In 1973, the Cosmetic Toiletry and Fragrance Association (CTFA) instituted microbial limits of 5 × 10²CFU/g in products for infants and the eye area and a maximum of 10³CFU/g for all other types of cosmetic products. Furthermore, no product can contain harmful microorganisms.⁴

Nearly all pharmaceutical formulations are likely to be contaminated with microorganisms.⁵ The growth of fungi, bacteria, yeasts and spores in products depends on a number of chemical and physical factors such as water content, nutrient availability and temperature. Non-sterile formulations generally have requirements for microbial growth including water, minerals, vitamins, oil and soluble polymers, and an environment with favourable oxygen content, pH and temperature.^6,7

The low quality of some non-sterile products might be due to contamination resulting from a lack of hygiene in manufacturing, inadequate filling conditions and the low stability of its constituents. It is important to avoid contamination during manufacturing, which is an environment with excellent conditions for microbial growth. Contamination can result in visible or non-visible modifications of the final product such as changes in colour, odour, viscosity, rheological properties, taste (for example, in mouthwash or oral rinses), bacterial visualisation and/or fungal colonies, and it also may induce a reaction in the user.²

Non-visible modifications that indicate microbial deterioration should be controlled, especially if the product is to be used in burn patients, in those with damage to the epithelium or in immunocompromised individuals.⁸ Contamination can result in the withdrawal of the product batch from the market, the cancellation of the registration of responsible companies and a lapse of credibility, resulting in huge losses for the companies.⁹

The raw materials of cosmetics, both hydrophilic and lipophilic, can provide nutrients for bacterial and fungal growth. Natural raw materials have many routes of contamination including air, water, soil, human handling and the processing area and can result in the presence of microorganism toxins and metabolites. Although more difficult to contaminate, synthetic raw materials can also be altered by decomposition caused by microorganisms.^9,10

According to Orth¹¹ some compounds can potentiate preservation while others impede it. Raw materials that can contribute to antimicrobial activity include alcohol (such as ethanol), polyols (such as glycerin and propylene glycol in concentrations above 20%) and antioxidants (such as sodium metabisulfite). Raw materials that hinder preservatives include an excess of metal ions such as Mg⁺² and Ca⁺², which react to form insoluble complexes and catalyse oxidative changes, and some non-ionic surfactants that compromise preservation through complex formation, interfering with the preservative solubility in the surfactant hydrophilic portion, oxygen bridge formation, and others.¹²Table 1 lists antimicrobial materials and their inactivating agents.

Table 1 Selected antimicrobial materials and their respective neutralising agents

Antimicrobial agents	Inactivating agents
Formaldehyde	0.1% histidine
Chlorine derivatives	0.5% Na2S2O3
Quaternary ammonium	3% polysorbate 80 + 0.3% lecithin
Amphoteric surfactants	3% polysorbate 80 + 0.3% lecithin
Phenol and derivatives	1% polysorbate 80
Heavy metals	0.1% cysteine
Isothiazolinones	12% sodium bisulfite

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The packaging, and especially any primary packaging that is in direct contact with the product, must be considered because, although sterility is not necessary, the presence of large quantities of microorganisms can contribute to microbial contamination in the product, which reduces its quality.¹³

In addition, it is necessary to show that the microorganisms present have metabolic versatility and may use preservatives as substrates. Close and Nielsen¹⁴ verified that Pseudomonas cepacia was able to utilise the microbial preservative propylparaben as a carbon source, hydrolysing it to methylparaben and thereby inactivating it.

Despite the described risks, some authors do not deem it necessary to use preservatives or perform challenge tests for products with or without a low amount of water or for products that are considered to have an inadequate environment for microbial growth including powder compacts, stick products and products with alcohol or waxes.^14,15

Corbett¹⁶ describes the most probable microorganisms related to various contamination sources including water (Pseudomonas, Xantomonas, Flavobacterium and Achromobacter), air (fungal spores, Penicillium, Mucor, Aspergillus, Bacillus sp., yeasts and bacterial spores), raw material (Clostridium sp., Salmonella sp., coliforms, Actinomyces, mould and yeast) and processing (coliforms, Staphylococcus, Streptococcus and Corynebacterium).

The action spectrum of potent preservative systems can be assessed primarily by in vitro studies. Some microorganisms can be studied, for example, by inoculation and microdilution plating techniques.⁹

The industry is adapting the few official regulations regarding microbial content in non-sterile products to those for pharmaceutical and food products. The publications of government and cosmetic associations only specify that products should not cause damage to human health in normal and predictable use conditions.⁷

The European Union Cosmetic Standard requires that all cosmetics present the microbiological information of the raw materials and finished products on the label as well as the criteria for microbiological control. The FDA recommends that all batches of non-sterile, non-self-preserved products be verified for microbiological contamination, and all cosmetic products should be analysed in the development phase for adequate preservation.⁹

The European and American Pharmacopoeias define “microbial preservatives” as substances that, when added to the non-sterile products, and particularly to those with an aqueous base and multiple-use packaging, preserve the products from damage caused by microorganisms during manufacturing, storage and accidental contamination by consumers during use.^17,18

Preservatives are biologically active chemical products that are added to avoid microbiological growth and proliferation. They can inhibit enzymes and block synthesis of proteins and nucleic acids, but they could also harm the consumer (allergy and toxicity). Therefore, they should be used in the lowest possible concentrations to guarantee product quality without harming the user.^8,15

Despite several types of available preservatives, few are used. However, they can be used in combination. These substances are added to formulations with two objectives. The first is to protect the user from harm due to contamination, and the second is to maintain the quality of the product, as contamination leads to drastic changes in their physical and chemical features.^1,2,8,19,20

Preservatives are added not only to reduce the microbial count and to satisfy the challenge test, but also to avoid microorganism growth and reproduction.¹⁵

Microbiological control ensures that the product is of good quality and free from certain microorganisms, particularly those that are harmful to the user, and it guarantees that adequate quality is maintained throughout continuous use by the consumer.¹

The aim of this work was to review the challenge test for the efficacy of preservatives in non-sterile products by focusing on the historical context, explanation of the technique, interpretation of the results according to pharmacopoeias and emphasising the critical points in the technique.

Historical context

Though several regulated preservatives are available, few are used in cosmetic and pharmaceutical products. It is often necessary to use them in combinations.²¹

The challenge test was developed to simulate preservative conditions and impose excessive abuse on the products to predict with accuracy the normal response to an accidental microbial exposure.¹⁵

Challenge tests and preservative effectiveness are included in product safety testing. Although aqueous cosmetic and pharmaceutical products and drugs packaged in multiple-use containers are not required to be sterile, they should have a suitable preservative system. Preservatives eliminate bacterial contaminants before they become dangerous to the consumer and ensure the physical quality of the product.⁹

A method to evaluate the preservation system was first described by the American Pharmacopoeia in 1970 and was only applicable to aqueous, sterile, injectable ophthalmic and nasal products. This method has remained unchanged for about twenty-five years due to its reproducibility, and it represents a significant contribution to the study of preservative effectiveness.⁴

Other publications, including those of the Federation Internationale Pharmaceutiques (FIP), the British Pharmacopoeia, in addition to The Cosmetic Toiletry and Fragrances Association (CTFA), reported methodologies with different interpretations.¹²

Methodology

The challenge test is usually recommended to study preservative systems and microbial stability. This test can be performed when the product is likely to have contamination that is potentially dangerous to the consumer.⁴ In the Guideline for the Safety Evaluation of Cosmetic Products, the ANVISA, a Brazilian Agency, recommends that this test be performed both in accelerated and long-term stability studies of a product. It should be performed at the beginning and end of the stability tests to analyse whether the conditions resulted in a loss of or ineffectiveness of the used preservative(s). Thus, this test can be used to determine if the types and concentrations of the preservatives are adequate.²²

The challenge test also determines the minimal effective concentration to guarantee that the product remains compliant with laws during its manufacturing, packaging, and distribution, in addition to preventing possible unintended contamination by the user.^23,24,25

Knowing the mechanism of the preservative system is necessary in order to perform the appropriate tests to analyse the preservative's potential based on the dilution in the culture medium and its ability to inhibit standard microbial strains. The active concentration interval or the unique lethality point is determined, and the incubation conditions are verified to define the biocidal or biostatic activity.⁹

In general, it is acceptable that the microbial counts in a non-sterile product remain within the official limits during the storage time (shelf life), but increases in microbial counts are not desirable.⁴

In this test, known as a “challenge”, 10⁵–10⁶CFU/mL of inoculum of the bacterial species Staphylococcus aureus, Pseudomonas aeruginosa or Escherichia coli or the fungal species Aspergillus niger and Candida albicans are added to the preservative or to formulations containing them, and changes in the appearance of the product and viability of these microorganisms over time are recorded.^18,26

D value

An alternative method to assess the efficacy of preservatives relies on the microorganism populations predictably losing their viability and the population number decreasing exponentially with time. The D value is defined as the time required for a 90% reduction (1 log) of the population when submitted to an agent under constant conditions. These values can be calculated with a curve expressed by a function of the log survivor number and the time (linear regression) after inoculation.^9,27

This unofficial method is widely used when studying degradation due to microorganisms and is based on the probability of survival through a sterilisation process, which is dependent on the initial microbial population and its inactivation kinetics. When a homogenous microorganism population is constantly exposed to a lethal process, it loses its viability predictably. The inactivation rate is directly proportional to the number of organisms present at a given moment. Therefore, a constant survivor proportion is inactivated for each exposure increment to the lethal agent. Mathematically, microorganism inactivation is described as a first order reaction.²⁸

The Orth method, which is similar to the method recommended by pharmacopoeias, applies the linearity concept to explain the reduction of time involved. As recommended by the author, the inoculum should be prepared in 0.1 mL of saline with product samples of 50 g. The samples should be inoculated separately to obtain approximately 10⁷CFU/g. The surviving population must be enumerated after 2, 4, 24 and 48 h for bacteria; 4, 8, 24 and 48 h for yeast; and 4, 8, 24, 48 and 7 days for mould.²⁸

This linear regression method has several advantages. The results are obtained within a few days, and it is possible to estimate the time required for the elimination of an entire microbial population in a product. Further, the acceptance criteria are based on quantitative measurements because the elimination of a specific microorganism in a given product can be used to study additive and synergistic effects of preservative combinations, which can indicate whether the packaging is adequate to prevent contamination. Contamination is less likely in products that meet the stricter requirements of this method.²⁹

Protocols from different pharmacopoeias

The efficacy test protocols and the interpretation of the results for preservative systems in the 1980s were similar to the described challenge test. Although the challenge test is only recommended at the industrial development stage of a product and is not mandatory, the differences in the interpretation criteria adopted by various pharmacopoeias may cause problems.

According to the Japanese Pharmacopoeia,³⁰ the microorganisms employed in the test should represent those that can be found in the environment in which the product is produced, used or stored.

Feels et al.³¹ used the challenge test to examine several products on the market for years and found that they did not meet the requirements of the British Pharmacopoeia.³² The authors stressed that the later formulations of these products were not altered, as this would increase the concentration of the preservatives beyond the permitted levels.

According to Orth¹¹ and Brannan³³ the microorganisms used in the test should represent Gram-positive bacterial species such as Staphylococcus aureus, Gram-negative bacteria with diverse metabolic capacities such as Pseudomonas aeruginosa, coliform group bacteria such as Escherichia coli, yeasts such as C. albicans and fungi such as A. niger. The American Pharmacopoeia¹⁸ requires that the test use these microorganisms, and the European and British Pharmacopoeias require the same except that E. coli need only be investigated in products for oral use.^17,26

Conventional challenge test

For the conventional challenge test, 10⁵–10⁶cells g⁻¹ (mL) are individually inoculated after growth of each microorganism (E. coli, S. aureus, P. aeruginosa, C. albicans and A. niger) in casein soy broth for 6 h at 36 ± 1 °C (bacteria) or Sabouraud broth at 26 ± 1 °C (fungi) in 10 or 20 g sample aliquots. Immediately following inoculation, 1 g samples are removed from the contaminated formulations (time 0), diluted in sterile saline to an appropriate concentration for colony counting and transferred to agar plates containing casein and soy Sabouraud agar. After incubation at 36 ± 1 °C for 24–48 h (bacteria) and 25 ± 1 °C for 5–7 days (fungi), colonies are counted and the values are expressed in CFU/mL (g). During the analysis, the samples are kept at room temperature with light.⁴ At defined time intervals (Table 2), samples are removed to count the surviving microorganisms (CFU/mL or g).

Table 2 Analysis intervals for the conventional challenge test in accordance with the official compendiums and product classifications^a

Product category	USP ^18	BP^26	EP ^17	JP ^30
a Category 1: parenterals and ophthalmic; Category 2: oral products; Category 3: topical products; Category 4: othologic products.^26 Category 1: parenterals and ophthalmic; Category 2: topical products; Category 4: oral products.^17 Category 1: injections, other parenterals including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles; Category 2: topically used products with aqueous bases or vehicles, non-sterile nasal products, and emulsions including those applied to mucous membranes; Category 3: oral products other than antacids with aqueous bases or vehicles; Category 4: antacids with aqueous bases.^18 Category 1: products with aqueous bases; Category 1A: injections and other parenterals including otic and ophthalmic products; Category 1B: topically used products applied to mucous surfaces including liquids for nasal instillation and inhalants; Category 1C: oral products except antacids with aqueous bases; Category 1D: antacids (including solid forms of antacids intended for aqueous constitution); Category 2: products made with non-aqueous bases.^30
1	Bacteria: 7, 14 and 28 days	Bacteria: 6 h, 24 h, 7 days and 28 days	Bacteria: 6 h, 24 h, 7 days and 28 days	Bacteria: 7, 14, 21 and 28 days
	Yeasts/moulds: 7, 14 and 28 days	Yeasts/moulds: 7, 14 and 28 days	Yeasts/moulds: 7, 14 and 28 days	Yeasts/moulds: 7, 14, 21 and 28 days
2	Bacteria: 14 and 28 days	Bacteria: 14 and 28 days	Bacteria: 2, 7, 14 and 28 days	Bacteria: 7, 14, 21 and 28 days
	Yeasts/moulds: 14 and 28 days	Yeasts/moulds: 14 and 28 days	Yeasts/moulds: 14 and 28 days	Yeasts/moulds: 7, 14, 21 and 28 days
3	Bacteria: 14 and 28 days	Bacteria: 48 h, 7 days, 14 days and 28 days	Bacteria: 14 and 28 days
	Yeasts/moulds: 14 and 28 days	Yeasts/moulds: 48 h, 7 days, 14 days and 28 days	Yeasts/moulds: 14 and 28 days
4	Bacteria: 14 and 28 days	Bacteria: 6 h, 24 h, 7 days, 14 days and 28 days
	Yeasts/moulds: 14 and 28 days	Yeasts/moulds: 6 h, 24 h, 7 days, 14 days and 28 days

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This methodology described by Pinto et al.⁴ is similar to that recommended by the British, European and American Pharmacopoeias^17,18,26 with some changes. For example, the analysis intervals recommended by the British Pharmacopoeia differ from the American (Table 2), and the European Pharmacopoeia recommends testing for E. coli only in oral-use products.

These organisations also describe the use of specific preservatives rather than performing dilutions to inactivate the preservative in formulations to avoid false negative results.^18,26

Other studies use Letheen broth, a non-selective medium that allows the growth of most strict and facultative aerobic bacterial species and contains substances such as lecithin and polysorbate, which inhibit the action of most chemical preservatives in cosmetics.^34,35

Calculating the D value

To calculate the D value, 10⁵–10⁶cells g⁻¹ (mL) of each of tested microorganism (E. coli, S. aureus, P. aeruginosa, C. albicans and A. niger) previously grown in casein soy broth for 6 h at 36 ± 1 °C (bacteria) or Sabouraud broth at 26 ± 1 °C (fungi) are inoculated individually in aliquots of product samples (usually 10 or 20 g). Immediately after inoculation, 1 g samples are removed from the contaminated formulations (time 0), diluted in sterile saline solution to an appropriate concentration for colony counting and transferred to agar plates containing casein and soy Sabouraud agar. After incubation at 36 ± 1 °C for 24–48 h (bacteria) and 25 ± 1 °C for 5–7 days (fungi), colonies are enumerated and the values are expressed in CFU/mL (g). During the analysis, the samples are kept at room temperature with light.⁴ At defined intervals (Table 2), samples are removed to count the surviving microorganisms (CFU/mL or g). The procedure is the same as the conventional challenge test, but the times at which samples are removed to count the surviving microorganisms are different (Table 2).

The British Pharmacopoeia²⁶ and the United States Pharmacopeia¹⁸ have equivalent methodologies and time analyses to test the D value.

The European Pharmacopoeia¹⁷ considers the D value methodology to be a tool to analyse the time necessary to eliminate microorganisms after stressors such as sterilisation, a method of preservation, but it does not specify analysis time periods.

The CTFA method is used internally by the Cosmetic, Toiletry and Fragrance American Association and recommends that S. aureus, P. aeruginosa, A. niger, yeast and bacilli spores be used. They suggest that the inoculum should be 10⁶CFU/g or mL, or 10⁴CFU/g or mL for products for the eye area. The analysis should be performed at 0, 7, 14, 21 and 28 days.^12,36

For this test, mixed-organism or pure microorganism cultures can be used. However, pure cultures are more appropriate because it is possible determine the death rates of each organism and their resistance profile to a certain formulation,¹¹ though a mixture may more accurately reflect the normal contamination profile of a product.³³

Table 2 presents the analysis intervals for the conventional challenge test according to the organisations classified by product types.

Interpretation of results according several pharmacopoeias and CTFA

The CTFA requires a 3-log reduction of vegetative bacteria in 7 days and no increase until the end of the test period (28 days). For fungi, continuous reduction must be observed until the end of the test period.⁹

The interpretation of the conventional challenge test accepted by various official compendia is presented in Table 3. According to the D value methodology, there must be a 6-log reduction or total pathogen elimination in 4 h or less. For non-pathogenic vegetative bacteria, fungi and yeasts, there must be total elimination in 28 h or less.⁹

Table 3 Challenge test interpretation criteria for the conventional method, in accordance to the official compendiums^a

Product category	USP ^18	BP^26	EP ^17	JP ^30
a Category 1: parenterals and ophthalmic; Category 2: oral products; Category 3: topical products; Category 4: othologic products.^26 Category 1: parenterals and ophthalmic; Category 2: topical products; Category 4: oral products.^17 Category 1: injections, other parenterals including emulsions, otic products, sterile nasal products, and ophthalmic products made with aqueous bases or vehicles; Category 2: topically used products with aqueous bases or vehicles, non-sterile nasal products, and emulsions including those applied to mucous membranes; Category 3: oral products other than antacids with aqueous bases or vehicles; Category 4: antacids with aqueous bases.^18 Category 1: products with aqueous bases; Category 1A: injections and other parenterals including otic and ophthalmic products; Category 1B: topically used products applied to mucous surfaces including liquids for nasal instillation and inhalants; Category 1C: oral products except antacids with aqueous bases; Category 1D: antacids (including solid forms of antacids intended for aqueous constitution); Category 2: products made with non-aqueous bases.^30
1	Bacteria: not less than 1.0 log reduction from the initial calculated amount in 7 days, not less than 3.0 log reduction from the initial amounting 14 days, and no increase from the 14 days' amount at 28 days;	Bacteria: not less than 2.0 log reduction from the initial calculated amount in 6 h, not less than 3.0 log reduction from the initial amount in 24 h and no increase from the initial calculated amount in 28 days.	Bacteria: not less than 2.0 log reduction from the initial calculated amount in 6 h, not less than 3.0 log reduction from the initial amount in 24 h and no increase from the initial calculated amount in 28 day.	1A: Bacteria: 0.1% of inoculum amount or less in 14 days, and same or lower than level on the 14–28 days.
				Yeast and moulds: same or less than inoculum amount in 14 and 28 days.
				1B: Bacteria: 1% of inoculum amount or less in 14 days, and same or lower than level on the 14– 28 days.
				Yeast and moulds: same or less than inoculum amount in 14 and 28 days.
	Yeast and moulds: no increase from the initial calculated amount in 7, 14 and 28 days.	Yeast and moulds: not less than 2.0 log reduction from the initial calculated amount in 7 days, and no increase from the initial calculated amount in 28 day.	Yeast and moulds: not less than 2.0 log reduction from the initial calculated amount in 7 days, and no increase from the initial calculated amount in 28 day.	1C: Bacteria: 10% of inoculum amount or less in 14 days, and same or lower than level on the 14–28 days.
				Yeast and moulds: same or less than inoculum amount in 14 and 28 days.
				1D: Bacteria: same or less than inoculum amount in the 14 and 28 days.
				Yeast and moulds: same or less than inoculum amount in 14 and 28 days.
2	Bacteria: not less than 2.0 log reduction from the initial amount in 14 days and no increase from the 14 days' amount in 28 days.	Bacteria: not less than 3.0 log reduction from the initial calculated amount in 14 days and no increase from the initial calculated amount in 28 days.	Bacteria: not less than 2.0 log reduction from the initial calculated amount in 2 days, not less than 3.0 log reduction from the initial amount in 7 days and no increase from the initial calculated amount in 28 days.	Bacteria: same or less than inoculum amount in the 14 and 28 days.
	Yeast and moulds: no increase from the initial calculated amount in 14 and 28 days.	Yeast and moulds: not less than 1.0 log reduction from the initial calculated amount in 28 days.	Yeast and moulds: not less than 2.0 log reduction from the initial calculated amount in 14 days, and no increase from the initial calculated amount in 28 days.	Yeast and moulds: same or less than inoculum amount in 14 and 28 days.
3	Bacteria: not less than 1.0 log reduction from the initial amount at 14 days, and no increase on the 14–28 days.	Bacteria: not less than 2.0 log reduction from the initial calculated amount in 48 h, not less than 3.0 log reduction from the initial amount in 7 days and no increase from the initial calculated amount in 28 days.	Bacteria: not less than 3.0 log reduction from the initial calculated amount in 14 days and no increase from the initial calculated amount in 28 days.
	Yeast and moulds: no increase from the initial calculated amount in 14 and 28 days.	Yeast and moulds: not less than 2.0 log reduction from the initial calculated amount in 14 days, and no increase from the initial calculated amount in 28 day.	Yeast and moulds: not less than 1.0 log reduction from the initial calculated amount in 14 days, and no increase from the initial calculated amount in 28 days.
4	Bacteria: no increase from the initial calculated count at 14 and 28 days.	Bacteria: not less than 2.0 log reduction from the initial calculated amount in 6 h, not less than 3.0 log reduction from the initial amount in 24 h and no increase from the initial calculated amount in 28 days.
	Yeast and moulds: no increase from the initial calculated count at 14 and 28 days.	Yeast and moulds: not less than 2.0 log reduction from the initial calculated amount in 7 days, and no increase from the initial calculated amount in 28 days.

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Critical points

Due to the lack of regulation for the challenge test, many adaptations are used. For example, in a study by Hugbo et al.³⁷ 20 g of sample were contaminated with 1 mL of a suspension of microorganisms that are frequently found in non-sterile products (S. aureus, A. fumigatus and Penicillium spp.) rather than using 10⁵–10⁶CFU/mL as recommended by the official agencies. In addition, the analysis intervals were 1, 2, 6, 12 and 30 days, which is different than the recommendation.

None of the methodologies for the conventional test require the elimination of all microorganisms initially inoculated in the sample until the end of the analysis.⁹ This can allow the development and reproduction of the bacteria adapted to the stress conditions, contributing to contamination of the product.

The methodology proposed by the CTFA is more rigorous than the various pharmacopoeias for the interpretation of the bacterial results because it demands the elimination of more bacteria in a shorter period of time. The recommendations regarding fungi do not specify the necessary reductions or product classifications.

The D value method is more demanding because it requires total elimination of the inoculated pathogenic microorganisms in 24 h and of the non-pathogenic species in 7 days.⁹ This requirement improves the quality of the products, guaranteeing their stability. The European Pharmacopoeia¹⁷ does not suggest analysis intervals for the D value assay and none of the consulted pharmacopoeias specify the necessary reduction of microorganisms in relation to exposure time to the preservative.

The challenge test protocol does not clarify how to carry out the dilutions. For example, they do not specify whether culture media or saline solution should be used and how many dilutions are necessary to achieve an appropriate dilution for enumerating CFU/mL or g. Additionally, the aliquot volume that must be transferred to the plate is not given.

Due to the lack of information on how to perform the technique, researchers make changes to the technique by analysing existing data. The use of saline solution to dilute the microorganisms is preferable because it does not inactivate preservatives, as is the case with Letheen broth. Letheen broth is more expensive than saline and allows growth of the microbes. Generally, 1.0 mL dilutions are used because the result is given as CFU/mL or g of product.

The pharmacopoeias do not provide an explanation of how to analyse the results of the conventional method. Instead, they provide only acceptance criteria, thus making the interpretation of the results difficult. While available literature stresses the necessity of the challenge test and its protocols, explanations of the calculations involved are lacking. Further, Brazilian organisations only provide the Guide for Stability Studies of Cosmetic Products, which again stresses the importance of the challenge test but also fails to clarify the interpretation of the results.²²

However, more literature is available regarding the calculation of the D value. Although this technique is not officially recognised, it adequately determines the effectiveness of preservatives.

Calculations

Mazzola et al.³⁸ determined the disinfectant D value by inoculating 1 mL of microorganism suspension in 100 mL of disinfectant solution. In consistent intervals, 1 mL aliquots were transferred to 8 mL of culture medium containing a neutralising agent.

Hugbo et al.³⁷ do not present calculations for the analysis of their results. Rather, only the amount and type of microorganisms in the analysed products are described.

The D value is determined using a survival curve of the logarithmic reduction (log CFU/mL or g) of microorganisms as a function of the exposure time in minutes. When linear regression is performed, the minimum squares allow the inclination value determination and its reliable limits to determine the D value.⁴

The D value is useful because the probability of microorganism survival in a specific preservation depends on the initial microorganism population in the product (bioburden or biomass) and the inactivation kinetics when exposed to the lethal process.⁴

A hypothetical example of a D value calculation is presented. Two products (A and B) were analysed; product A contained a preservative and product B was identical except that it did not contain the preservative.

The analysis was carried out as described above (Methodology of D Value) and the obtained CFU/g log values of P. aeruginosa are described in Table 4

Table 4 Log CFU/g of P. aeruginosa, in products A and B, in function of time^a

Preservative system	0 h	2 h	6 h	12 h	24 h	48 h	7 d	14 d	21 d	28 d
a A = product with preservative; B = product without preservative; ND = no increase from the initial.
A	6.0	5.3	4.6	3.9	0	0	0	0	0	0
B	6.0	5.7	5.2	4.6	3.8	0	0	0	0	0
Control	6.0	ND	ND	ND	ND	ND	ND	ND	ND	ND

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The graphs for product A (Fig. 1) and product B (Fig. 2) were produced from the obtained results. Table 5 presents the equation, correlation coefficient and D values for products A and B using P. aeruginosa. In this fictitious example, the two analysed products would not be approved by the challenge test because the D values are greater than 4.0 h.

Fig. 1 Lethality profile – log CFU/g × time (hours) of P. aeruginosa in Product A.

Fig. 2 Lethality profile – log CFU/g × time (hours) of P. aeruginosa in Product B.

Table 5 Equation, correlation coefficient and D value for products A and B^a

Product	Equation	Correlation coefficient	D value
a A = product with preservative; B= product without preservative.
A	−0.2394x + 6.0665	0.98	4.1
B	0.1203x + 6.0616	0.99	8.2

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Analysis periods

The analysis periods proposed by the British Pharmacopoeia are more relevant than those of the American Pharmacopoeia because they allow more inferences on the behaviour of the preservatives during the first hours of incubation with microbes.

However, it is of interest to add intervals such as 2 h, 12 h and 21 days. More time points in the first 48 h of the assay would allow better analysis of the activity of preservatives, and analyses at 21 days would provide knowledge of the resistant microorganisms. These modifications can give insight into the preservative activity profile.

Thus, some researchers adapt the methods of the official agencies to meet the methodology specifications. For example, Chorilli et al.³⁵ modified the sample collection times for the D value calculations to 2, 24 and 48 h at room temperature to enumerate bacteria and yeast and 2 h, 24 h, 48 h and 7 days for moulds.

The McFarland scale

The McFarland scale consists of barium sulfate (BaSO₄) solutions that can be commercially acquired or prepared in the laboratory by adding 0.5 mL of BaCl₂ (0.048 mol L⁻¹; 1.175% w/v BaCl₂·2H₂O) to 99.5 mL of H₂SO₄ (0.18 mol L⁻¹; 0.36 N; 1% v/v). The turbidity of this mixture is verified using a 625 nm wavelength in a spectrophotometer. McFarland standards correspond to predetermined absorbencies; for the 0.5 standard, the absorbance must be from 0.08 to 0.10.³⁹

Approximately 6 mL of the standard are transferred to assay tubes and stored in the dark at room temperature for up to 3 months. This solution must be vortexed before use.³⁹

The 0.5 McFarland standard is used in both the conventional and D value methods to standardise the inoculum used in the challenge test. The standard and the prepared microorganism inoculation solution (in saline) are visually compared for similar turbidity.⁴⁰

It is necessary that the tubes be identical to those used for the McFarland standard because differences in thickness or composition can complicate the suspension standardisation.

This visual comparison is subjective and vulnerable to errors, mainly human errors, when performing this technique. Incorrect techniques and interpretations decrease the test accuracy as it is desirable to inoculate 10⁵–10⁶CFU/mL of microorganisms, and a different amount could be inoculated.

Conclusions

Importantly, the preservatives do not have to be used to substitute Good Manufacturing Practices (GMP), but they must hinder product microbial contamination in order to guarantee effectiveness, security and stability.⁷

In addition to being useful to inform manufacturers about the type and amount of preservative to be added to a formulation to assure its efficacy, the challenge test can also be used to verify if packaging appropriately protects the product from microbial contamination.^19,41

This test is included in safety and stability assays performed during the development of a product, but generally is not carried through in the routine control of the products.^21,42

Although there is no consensus regarding its use, the challenge test is the most adequate system to verify preservative effectiveness.

Acknowledgements

The authors would like to thank CNPq (Brasília, Brazil), FUNDUNESP (São Paulo, Brazil), FAPESP (São Paulo, Brazil) and PADC-FCF (Araraquara, Brazil) for their financial support to the projects under development.

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