Regulatory risk assessments: Is there a need to reduce uncertainty and enhance robustness? Update on propylparaben in relation to its EU regulatory status

Abstract

Over 10 years ago, propylparaben (propyl–p–hydroxybenzoate; PP) was withdrawn as a permitted food preservative in the EU based entirely on findings reported in a single dietary study in juvenile rats claiming to show adverse effects on male reproductive parameters [Oishi S. Effects of propyl paraben on the male reproductive system. Food Chem Toxicol 2002; 40(12): 1807 -1813]. Subsequent data reviews have cast serious doubt on the validity of the Oishi results, mainly in relation to aberrant concurrent-control values, and in two further comprehensive studies using neonatal and juvenile rats there were no adverse effects in males at oral doses up to 1000 mg/kg/day. By contrast, juvenile animal toxicity data on the two paraben preservatives currently permitted in the EU as food additives (methylparaben and ethylparaben) are non-robust and rudimentary. Although PP is a permitted preservative in cosmetics its use pattern is highly restricted based mainly on the results of a screening study in the rat using butylparaben as test material, and not taking into account the more recent data on PP. The European Medicines Agency has determined a permitted daily exposure of 2 mg/kg for PP, which applies to both adult and paediatric patients, based on an oral no-observed-adverse-effect level of 100 mg/kg/day in females, treatment-related changes suggestive of an estrogenic effect being noted at 1000 mg/kg/day. The weight of evidence strongly supports a toxicological re-evaluation of PP regarding its use in foodstuffs and cosmetics in the EU, with a view to reinstatement as a food additive, consistent with its status in other major jurisdictions.

Keywords

Propylparaben reproductive toxicity food additive preservative pharmaceutical excipient

Introduction

Parabens, alkyl esters of p-hydroxybenzoic acid, have been employed successfully since the 1920s as effective preservatives in foodstuffs, cosmetics and pharmaceuticals. Apart from propylparaben (PP), other commonly used parabens are methylparaben (MP), ethylparaben (EP) and butylparaben (BP) in which the n-propyl group in PP is replaced by methyl, ethyl and n-butyl, respectively. For the last 10 years or so, PP has been effectively banned as a food preservative in the European Union (EU)^1,2 (based on an assessment by an expert panel of the European Food Safety Authority (EFSA)), and its use in cosmetics is quite severely restricted.^3,4 In 2007, the Joint Expert Committee on Food Additives (JECFA), on almost identical grounds to those articulated by EFSA, recommended withdrawal of the acceptable daily intake (ADI) for PP⁵ – previously evaluated on the basis of a joint ADI of 0–10 mg/kg/day for MP, EP and PP. On the other hand, PP has been affirmed as generally recognized as safe (GRAS) in the United States with a maximum concentration of 0.1% in foodstuffs.^2,6 (Similar GRAS provisions apply to MP,⁷ but not EP.) In a previous publication,⁸ a case was made for a re-evaluation of PP (n-propyl-p-hydroxybenzoate; E216; CAS no 94-13-3) particularly in relation to its use as a food additive in the EU. An alternative form is sodium propyl p-hydroxybenzoate; E217; CAS no 35285-69-9. (There was an error in the PP structure shown previously;⁷ the correct structure is shown in Figure 1.) This article is intended to provide an update on PP based mainly on the reflection paper prepared by the Safety Working Party⁹ of the Committee for Medicinal Products for Human Use, the principal expert committee of the European Medicines Agency (EMA).

Figure 1.

Propylparaben.

Scientific and regulatory background

In 2001–2004, a Japanese university researcher (Oishi) published a series of single-author papers on MP, EP, PP and BP; the researcher reported potential adverse effects on male reproduction in juvenile Wistar rats at high oral dietary doses of PP and BP, but not MP or EP. The key 4-week study (Oishi¹⁰) on PP employed concentrations of 0.01%, 0.1% and 1.0% in the diet of male juvenile rats (eight animals/group), commencing shortly after weaning when the animals weighed about 50 g and were 19–21 days of age (postnatal day (PND) 19–21).⁹ The parameters examined were body weight, weights of the male sexual organs, sperm count in the cauda epididymis, sperm count in the testes and testosterone concentration in the blood serum. The dietary concentrations correspond to body weight–related doses of 10, 100 and 1000 mg/kg/day at approximately the midpoint of the study. At the start of the study, the food intake data indicate that the 50 g rats were receiving closer to 20, 200 or 2000 mg/kg/day as the initial daily dose.

A reduction in body weight of statistical significance was found at the highest dose and appears to be associated with an unexplained reduction in food intake occurring around day 22 of the study. None of the male sexual organs showed any decrease in absolute or relative weight that reached statistical significance at any dose, no histopathological examination being undertaken on any tissues.

The principal potentially adverse effects were as follows:

Serum testosterone: A statistically significant difference from control values was achieved at the 1000 mg/kg dose although there was an apparent dose-related trend to reduction of circulating testosterone from means of 9.08 ng/mL in control rats to 8.20 ng/mL at 10 mg/kg PP and 7.17 ng/mL at 100 mg/kg PP.

Sperm counts in the cauda epididymis: These showed a trend towards a treatment-related decrease although the reduction for the 10 mg/kg dose did not reach statistical significance, mainly because there was considerable variability in the control group in which the sperm concentration was 1080 × 10⁶/g.

Daily sperm production (DSP) in the testes: This parameter revealed a 30% reduction at all dose levels (25.9–27.0 × 10⁶/g); however, there was no dose response, sperm counts being almost identical at all three dose levels; DSP in the concurrent control animals was 37.5 × 10⁶/g.

Historical control serum testosterone concentrations in neonatal/juvenile Wistar rats are up to 2.5 ng/mL. On the other hand, control means in the Oishi PP and BP studies were about 9 ng/mL.¹¹ For the two ‘positive’ studies of Oishi on PP and BP,¹² control epididymal sperm counts were well above the normal range (overall 108–1068; mean 561 ± 250 × 10⁶/g) established by the US National Toxicology Program,¹¹ and for the ‘negative’ studies on MP and EP,¹³ control epididymal counts (40 × 10⁶/g) were exceedingly low at around one-third of the lowest value for the normal range of historical controls.¹⁴ Control values for DSP/g × 10⁶ in the two other Oishi juvenile rat studies are 21.4 (MP/EP) and 25.4 (BP), indicating that the concurrent control value of 37.5 in the PP study is abnormally high which, in turn, would explain the lack of dose response in the animals treated with PP (for which DSP was in the range 25.9–27.0).

The mechanism of PP’s alleged adverse effects proposed by Oishi (direct effect on sperm and/or an indirect effect based on oestrogenic effects and/or testosterone reduction) is considered to lack plausibility. For example, after oral administration, PP is rapidly hydrolysed to p-hydroxybenzoic acid¹⁵ and the spermicidal concentration of 3.0 mg/mL for intact PP reported by Song et al.¹⁶ would never be achieved in vivo.

In summary, the proposed mechanism of action of PP is not supported by the data; outlier concurrent control values are apparent for DSP, epididymal sperm counts and testosterone and are not consistent with literature data (and data from other studies of Oishi), and a dose response is lacking for DSP.

Having been alerted by industry to the deficiencies in the Oishi publication on PP, the Scientific Committee on Consumer Products (SCCP)¹⁴ initiated a formal request by the European Commission for access to the full protocols and raw data of the Oishi publications but was informed that these were ‘no longer available’ (for the studies on MP and EP as well as on PP, see Tables 1 and 2). In the same document, SCCP concluded that a publication by Hoberman et al²³ designed to evaluate the same endpoints as the Oishi study but using MP and BP as test materials did not provide an unarguable refutation of the Oishi findings.

Table 1.

Toxicity studies in neonatal/juvenile rats relating to regulatory risk assessment of PP.

Study; test material	Design conditions	GLP	TK	Raw data audit	NOAEL (mg/kg/day)	Comment (see text)	Risk assessment supported
Oishi¹⁰; PP	PND 19–21 males; dietary doses of ca 10, 100, 1000 mg/kg/day for 4 weeks; no histopathology	N	N	Data ‘not available’¹⁴	≤10	Claimed effects on sperm production and testosterone levels. Refuted study; aberrant concurrent control data	EFSA/JECFA food additive
Fisher et al.³⁰; BP	PND 2 males; SC dose of 2 mg/kg/day for 17 days; no histopathology	N	N	N	2.0	NAE. Single-dose screening study; limited endpoints	SCCS cosmetics for PP (and BP)
Vo et al.¹⁸; PP	PND 21 females; oral gavage doses of 62.5, 250 and 1000 mg/kg/day for 20 days; no histopathology	N	N	N	[250]¹⁹	SCCS: ‘LOAEL cannot be derived. Recent toxicokinetic data indicate low systemic exposure to BuPB even at high doses and raise doubt on the relevance of the study’³	PDE of 5 mg/kg/day for PP as a pharmaceutical excipient in adults and metabolically mature children (EMA draft reflection paper¹⁹)
Gazin et al.²⁰; PP	PND 21 males; oral gavage doses of 3, 10, 100, 1000 mg/kg/day for 8 weeks; histopathology; reversibility	Y	Y	Y	1000	NAE. ‘Comprehensive study; statistically robust’; NOAEL confirmed by SCCS³ and EMA⁹	Not used for any PP risk assessment
Pouliot²¹; PP	PND 4–90 males and females at oral gavage doses of 10, 100, 1000 mg/kg/day; histopathology, reversibility	Y	Y	Y	1000 (males); 100 (females)	Confirms Gazin et al NOAEL for males. Possible estrogenic effect in females at 1000 mg/kg/day⁹.	PDE of 2 mg/kg/day as a pharmaceutical excipient in adult and paediatric patients (EMA reflection paper⁹)

BP: butylparaben; EFSA: European Food Safety Authority; EMA: European Medicines Agency; GLP: Good Laboratory Practice; JECFA: Joint WHO/FAO Expert Committee on Food Additives; N: no; NAE: no adverse effects; NOAEL: no-observed-adverse-effect level; PP: propylparaben; SCCS: Scientific Committee on Consumer Safety; TK: toxicokinetics; Y: yes; PDE: permitted daily exposure.

Table 2.

Toxicity studies in juvenile rats relating to regulatory risk assessments of MP and EP.

Study; test material	Design conditions	GLP	TK	Raw data audit	NOAEL (mg/kg/day)	Comment	Risk assessment supported
Oishi¹³; MP	PND 25–27 males; dietary doses of ca 100, 1000 mg/kg/day for 8 weeks; no histopathology (apart from testis)	N	N	Data ‘not available’¹⁴	1000	No effects on bodyweight gain, organ weights, serum hormones and sperm counts; study quality could not be properly assessed²²	EFSA/JECFA/EMA
Hoberman et al.²³; MP	PND 22 males; dietary doses of ca 10, 100, 1000 mg/kg/day for 8 weeks;	Y	N	Y	1000	Endpoints as above plus histopathology on reproductive tissues. SCCS critical of high variability of some key parameters²²	Not used for any MP risk assessment
Oishi¹³; EP	PND 25–27 males; dietary doses of ca 100, 1000 mg/kg/day for 8 weeks; no histopathology (apart from testis)	N	N	Data ‘not available’¹⁴	1000	No effects on bodyweight gain, organ weights, serum hormones and sperm counts; study quality could not be properly assessed²²	EFSA/JECFA/EMA

EFSA: European Food Safety Authority; EMA: European Medicines Agency; EP: ethylparaben; GLP: Good Laboratory Practice; JECFA: Joint WHO/FAO Expert Committee on Food Additives; MP: methylparaben; N: no; NOAEL: no-observed-adverse-effect level; SCCP: Scientific Committee on Consumer Products; TK: toxicokinetics; Y: yes.

In the most recent EU review of PP in 2013,³ the Scientific Committee on Consumer Safety (SCCS) evaluated an 8-week oral-gavage toxicity study in juvenile rats (PND 21–77) on PP by Gazin et al.²⁰ (which includes a significant toxicokinetic component) that was designed to evaluate the same parameters investigated by Oishi. No adverse effects were noted, and a no-observed-adverse-effect level (NOAEL) at the highest dose of 1000 mg/kg/day was determined. SCCS concluded that

The GLP (Good Laboratory Practice) study on reproductive toxicity (of PP) has been well conducted and is considered appropriate to refute the study of Oishi which reported reproductive toxicity in juvenile male rats. The toxicokinetic data indicate a rapid and effective metabolism of propylparaben after oral exposure due to rapid and effective hydrolysis of the substance by carboxylesterases.

However, SCCS commented that the study does not cover the potentially sensitive period after birth until PND 21 and also questioned its relevance for human risk assessment of dermally administered PP.

PP: EMA reflection paper

The EMA reflection paper⁹ mentioned above, released at the end of 2015, contains summary information from a new study on PP – designated as Pouliot, 2013.²¹ (The results of this study are available in a 2014 publication.²⁴) This study was of similar design to the Oishi, Hoberman and Gazin studies and involved administration of PP to PND 4 male and female rats for 86 days; the results for male animals are summarized as follows in the EMA reflection paper:

A GLP-compliant study was conducted to determine the potential toxicity and reproductive effects of propylparaben in juvenile Sprague-Dawley rats (25 per sex per group) treated by oral gavage from PND 4 through 90 at 0 (vehicle), 10, 100, and 1000 mg/kg/day. Male animals were necropsied either at the end of treatment (10 per group), or at the end of a 40-day treatment-free period (15 per sex per group) used to evaluate their reproductive capability when mated with naïve females. In males, there were no compound-related effects on organ weights, no gross or histopathologic findings in reproductive tissues, no effect on sexual maturation and no evidence of an effect on the reproductive capability (mating and fertility indices, number of days to mating, conception rate). Toxicokinetic investigations were conducted on PND 7, 21 and 83. Exposure levels to propylparaben and three of its metabolites (p-hydroxybenzoic acid, sulfate of propylparaben, sulfate of p-hydroxybenzoic acid) were greater in 7-day-old rats and decreased with increasing age. The duration of exposure between dosing intervals was inversely correlated to the age, in line with ongoing metabolic maturation from 7 days to 83 days of age. In the highest dose group, nonconjugated propylparaben was detected up to 24 h, 8 h and 8 h after dosing on PND 7, 21, and 83, respectively. At the same time, metabolites were detected up to 24 h, 24 h and 8 hours, respectively, after dosing on PND 7, 21, and 83, respectively. In all dose groups and age subsets, propylparaben was a minor circulating analyte with mean systemic exposures that were less than 1% of total measured AUC (area under curve). Overall, there were no propylparaben-related changes on a range of endpoints that could be suggestive of an estrogenic effect, and the dose of 1000 mg/kg/day was the no-observed effect level in males (Pouliot, 2013). This study confirms the results obtained by Gazin et al (2013) and even extend them since rats were dosed from the neonatal period. It also strengthens the NOEL value of 1000 mg/kg/day of propylparaben for juvenile male rats.

The results for treated PND 4 females are shown below:

Female animals were necropsied either at the end of treatment (10 per group), or at the end of a treatment-free period (15 per sex per group) used to evaluate any effect on their reproductive capability when mated with untreated proven breeder males. In the latter case, females and their pups were sacrificed 5 to 7 days post-partum. Most parameters investigated remained unaffected by treatment with propylparaben at any dose level, notably: histology of reproductive tissues, oestrous cyclicity, mating and fertility, maternal performance, and parameters evaluated on pups (birth weight, litter size, viability, clinical observations, external malformations). However, significant effects were observed on the onset of puberty (accelerated) and on the weight of uterus (increased) at 1000 mg/kg/day. Toxicokinetic parameters measured in females were not significantly different from those measured in males (as shown above). The exception was that 21-day-old females were exposed to non-conjugated propylparaben for a longer duration after dosing on PND 21 (detected up to 24 h after dosing at 1000 mg/kg/d). Overall, the dose of 100 mg/kg/day was the no-observed effect level in females since propylparaben-related changes suggestive of an estrogenic effect were observed at 1000 mg/kg/day.

This new study adds to the four previous ones utilizable for PP risk assessment (Table 1). Using the NOAEL of 100 mg/kg/day in female juvenile rats, a permitted daily exposure (PDE) of 2 mg/kg was calculated for both adult and paediatric patients⁸ (based on methodology described in the International Council on Harmonisation guideline on residual solvents – ICH Q3C²⁵).

MP, EP and PP: Comparison of their toxicological evaluations

Prior to 2006, all three parabens (MP, EP, PP) used as food additives were considered to be largely similar in respect of their toxicological profiles with a group ADI of 10 mg/kg/day. JECFA set this group ADI in 1973,²⁶ and in 1994, the then EU expert committee (Scientific Committee for Food) established an identical temporary group ADI based on an overall NOAEL of 1000 mg/kg/day.²⁷ In a follow-up review in 2004,²⁸ the EFSA AFC (Scientific Panel on Food Additives, Processing Aids, and Materials in Contact with Food) considered that

propyl paraben should not be included in this group ADI because this specific paraben, unlike the methyl and ethyl forms, had effects on sperm production at a relatively low dose in male juvenile rats. The Panel was unable to recommend an ADI for propyl paraben because of the lack of a clear NOAEL for this effect.

This opinion was based entirely on the results of the study of Oishi¹⁰ in juvenile male rats (Table 1), as was the subsequent JECFA evaluation. At the time, the chair of the EFSA expert panel¹ commented that: ‘This issue could be reconsidered if further evidence becomes available’. However, this does not seem to have happened in relation to PP which is still not permitted as a food additive in the EU.

What about MP and EP: Is the same standard of evidence available as is now the case for PP? Based on the studies reported in juvenile animals for MP and EP (Table 2), it is clear that PP has been tested for potential adverse reproductive effects in juvenile animals much more extensively than MP or EP. In addition, SCCS was critical of various aspects of all three studies on MP and EP mentioned in Table 2: ‘The shortcomings of the Hoberman study (on MP) prevent its acceptance. It cannot be used to refute the Oishi findings; these, in turn, cannot be properly assessed due to the unavailability of raw data’.²² Furthermore, no toxicokinetic data are available on MP or EP. In spite of the much more thorough evaluation of PP compared to those for MP and EP, and the problems of data integrity/auditability, EMA commented: ‘based on the totality of the in vitro and in vivo data, it can be concluded that methylparaben seems to be devoid of adverse effects on reproduction and development’. For EP EMA cross-referred to the previous EFSA evaluation:

EFSA has established a full-group ADI of 0–10 mg/kg body weight for the sum of methyl and ethyl parabens and their sodium salts (Directive 2006/52/EC). This limit is considered applicable also for medicinal products and precludes the need for another (PDE) calculation based on ICH Q3C.⁹

Discussion and conclusions

Parabens have been successfully employed as effective preservatives in foodstuffs, drugs and cosmetics for many decades. Mixtures of parabens are thought to act synergistic in relation to increased antimicrobial activity, and the MP–PP combination, typically in a 10:1 w/w ratio, is used extensively in pharmaceutical products.² However, based on a kinetic evaluation of the microbiological efficacy of MP–PP combinations, the study of Gilliland et al.²⁹ concluded that

parabens are synergistic since in combination they produce an effect which is not observed when they are used singly. This effect is not true synergism as shown by the results of our experiments with a factorial design. Analysis of variance indicated no significant interaction between the two parabens.

Although toxicological data on MP–PP combinations appear to be lacking, there is no reason to believe that there would be any enhancement of toxicity since both compounds are readily hydrolysed by carboxylesterases and have for many decades been safely used and authorized (both being of GRAS status in the USA) for combination use.

Although the in vitro oestrogenic activity of the parabens is normally considered to increase in relation to the chain length of the alkyl ester moiety, examination of in vivo data cited in the EMA reflection paper⁹ does not confirm this perception in that MP has greater or essentially equivalent activity compared to PP in three out of four uterotrophic assays. Neither MP nor EP has been tested in neonatal animals (i.e. PND 4 rats as performed by Pouliot²¹ on PP). Moreover, all of the juvenile animal toxicity studies on MP and EP (Table 2) are considered to be deficient to a lesser or greater extent based on the inability to perform a raw data audit (on the Oishi studies) and other criticisms of the study of Hoberman et al.²³ (partly rebutted by Scialli¹¹). The European regulatory status of PP is distinctly inferior to those for MP and EP in relation to use as a food additive⁸ and its restricted use in cosmetics continues⁴ in spite of the fact that parabens are considered to be the least sensitizing preservatives in commercial use.¹⁷ The most recent SCCS evaluation of PP³ as a cosmetic preservative was released in October 2013 and does not take into account the data from the study of Pouliot.²¹ The toxicity metric employed by SCCS in relation to the safety of PP as an ingredient of cosmetics is obtained from a single-dose screening study³⁰ using subcutaneous (SC) administration of BP (not PP). Moreover, as noted by Scialli,¹¹ SC administration ‘bypasses the portal of entry hydrolysis in skin and gastrointestinal tract, allowing greater internal exposure to parent compound’.

Overall, in contrast to MP and EP, the status of PP as a food additive and a cosmetics preservative is considered to be inconsistent with the extent and quality of the available toxicological data. European regulatory concerns/restrictions on PP appear to be a legacy of the initial EFSA and JECFA assessments in which there was a failure to detect the anomalous concurrent control values on sperm counts in the study of Oishi (when compared to values reported by Oishi in the MP/EP studies and to historical control data). In 2004, Ashby et al.³¹ emphasized the need to take into account natural variability in concurrent control values when detecting and interpreting weak endocrine toxicities. Another key factor was the decision to withdraw PP’s ADI on the basis of a single study before data from any confirmatory study became available. A 2006 review of PP by the US Environmental Protection Agency³² articulated various criticisms of the Oishi study and concluded that ‘dietary concentrations of 0.01–1.0% for four weeks resulted in decreased sperm production and efficiency in young rats. However, these reproductive parameters have not been verified or replicated’. A more recent critique of the study of Oishi is provided in a European Public Maximum Residue Level Assessment Report³³ on PP:

The reliability of this study has been discussed and questioned. The main arguments have been the lack of raw data, no data on systemic exposure and signs of lack of appropriateness for evaluation of relevant parameters, indicated by standard deviations which were far less than expected based on normal biological variability, and mean values for some parameters far outside accepted historical control ranges.

This report goes on to discuss the study by Gazin et al.²⁰ in juvenile male rats and comments as follows in relation to the 2004 evaluation of PP by EFSA: ‘If the new (2012) GLP-study in which a NOEL of 1000 mg/kg bw/day was established had been available at that time, propyl 4-hydroxybenzoate and its sodium salt would quite possibly have been included in that group ADI.

Information in Tables 1 and 2 indicates that the key studies supporting the safety of PP in juvenile animals (particularly those by Gazin et al.²⁰ and Pouliot²¹) are GLP-compliant. On the other hand, the two studies (Oishi¹⁰ and Fisher et al.³⁰) employed in evaluations by EU food/cosmetics regulatory bodies are non-GLP. Although lack of GLP compliance does not in itself diminish the scientific reliability of these studies, the GLP status of the studies of Gazin and Pouliot, combined with the availability of auditable raw data, is considered to increase the weight of evidence favouring the use of data from the latter studies for risk assessment purposes.

In view of the above discussion, and the absence of adverse effects on sperm counts in juvenile animals noted in several more recent comprehensive studies, it would not be unfair to claim that the current EU assessments, particularly in relation to the use of PP as a food additive, are compromised. In conclusion, surely the time is right for an EU reassessment of PP?

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflict of interest with respect to the research, authorship and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship and/or publication of this article.

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