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Para-hydroxybenzoic acid (4-HBA) was assessed for its estrogenic activity. A valid and reliable study with repeated administration of oral dosages up to and including 1000 mg/kg bw using rats (42 days) and valid and reliable studies in immature female rats and mice with repeated administration of dosages up to and including 100 mg/kg bw for 3 consecutive days (uterotrophic assays) were available and mainly used for assessment.
The repeated dose toxicity study in rats (Nagao et al., 1997) did not indicate any effects on reproductive organs of female and male rats or/and functional aspects of fertility by p-hydroxybenzoic acid. The reliable uterotrophic assays (Twomey 2000b, Hossaini et al. 2000) were negative for p-hydroxyenzoic acid in rats and mice in concentrations up to and including 100 mg/kg bw.
In summary there are a sufficient number of sufficiently reliable and valid studies for the evaluation of possible estrogenic effects of p-hydroxybenzoic acid. None of these studies showed an estrogenic effect of p-hydroxybenzoic acid. Hence, it is concluded that p-hydroxybenzoic acid has no estrogenic activity.

Additional information

According to REACH Art. 57 endocrine active substances sometimes also named as endocrine disruptors are Substances of Very High Concern.

Therefore it is necessary to assess p-hydroxybenzoic acid with respect to its endocrine properties, i.e. with respect to its estrogenic activity.This assessment was based on the following endpoints:

- in vitro studies: (i.e. OECD guideline 455, describing two approved in vitro stably transfected transactivation assays in cell lines, namely the STTA assay using the ERalpha-HeLa-9903 cell line and the BG1Luc ER TA assay using the BG1Luc-4E2 cell line (adopted in 2012))

- in vivo: repeated dose toxicity studies with investigations on reproductive organs and functional aspects of fertility (i.e.: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test))

- in vivo: specific studies (i.e. OECD guideline 440, the in vivo uterotrophic bioassay in rodents (of the year 2000))

There are no sufficiently reliable in vitro studies available.

 

A valid and reliable repeated dose toxicity study with oral administration by stomach tube of dosages of 40, 200 and 1000 mg/kg/d of p-hydroxybenzoic acid in male and female rats with 13 animals per group (Nagao et al., 1997) is available. The administration to the males was 42 days, the administration to the females was all in all between 38 to 52 days depending on the mating period.

The doses in this study were selected from the results of a preceding preliminary test with 14 -day repeated oral administration. With administration of 1000 mg/kg bw in male animals and 250 mg/kg bw or more in female animals, tendencies toward suppressed body weight gain were observed. Also, a slight reduction in food consumption was observed in female animals with doses of 1000 mg/kg bw. However, from the fact that no pronounced changes in food consumption, urinalysis, haematology tests, blood biochemistry tests, organ weight or autopsies were observed during administration, it was concluded that 1000 mg/kg bw was an amount less than the maximum tolerated dose and was set as the maximum dose for the combined test. Below this, with a common factor of 5, an intermediate dose of 200 mg/kg bw and a minimum dose of 40 mg/kg bw were established. This dose selection is in line with the general rules of toxicology: the lowest dose should be a no effect dose, the highest dose should show some toxicity without lethal effects during the study period. The observed changes were not considered as adverse effects. Thus, the NOAEL for systemic toxicity was considered to be 1,000 mg/kg bw/day, which complies with the evaluation of OECD SIDS(1999).

 

Within the study, reproductive organs in male and female rats (testes, epididymis, ovaries and uterus) as well as functional aspects on fertility (spermatogenesis, copulation index, fertility index, estrus cycles), which are assumed to be possibly affected by estrogens, were investigated. With these parameters no significant differences between p-hydroxybenzoic acid treated animals and controls were observed. Hence, results of the reliable in vivo repeated dose-toxicity study in rats did not give any hint on an estrogenic activity of p-hydroxybenzoic acid.

 

According to the `Guidance on information requirements and chemical safety assessment Chapter R.7a: Endpoint specific guidance´ (ECHA 2015) the uterotrophic assay according to EU B.54, OECD TG 440 in rodents (a short-term screening test for oestrogenic/ anti oestrogenic properties) is accepted by ECHA as one of the available in vivo assays focusing on identification of endocrine disrupting potency.

 

Specific studies (uterotrophic assays) with repeated administration of dosages up to and including 100 mg/kg bw for 3 consecutive days are available and mainly used for assessment. The reliable uterotrophic assays (Twomey 2000b, Hossaini et al. 2000) are negative for p-hydroxybenzoic acid in rats and mice in concentrations up to and including 100 mg/kg/d. Hence, it is concluded, that p-hydroxybenzoic acid has no estrogenic activity.

 

Discussions on endocrine disrupting effects of 4-HBA were started by a study of Lemini et al. 1997 with positive estrogenic effects according to the authors. At the highest dosage of 5 mg/kg bw increased weights of the uteri were reported by the authors. In 2003 the same authors published a further study with p-hydroxybenzoic acid. Here, the mice seemed to show estrogenic effects after repeated subcutaneous dosages of 150 mg/kg bw but not after 50 mg/kg bw. Because of the inconsistent results both studies and an additionally study with rats are considered as not reliable.

 

In 2013, the Scientific Committee on Consumer Safety (SCCS) published an updated scientific ‘Opinion’ (SCCS/1514/13) mainly related to endocrine disruptive properties of propyl- and butylparaben. In this Opinion it was stated (p.6) that “…p-hydroxybenzoic acid (PHBA)…is considered to be an inactive metabolite” related to its endocrine disruptive properties.

 

In summary there are a sufficient number of reliable and valid studies for the evaluation of possible estrogenic effects of p-hydroxybenzoic acid. None of these studies showed an estrogenic effect of p-hydroxybenzoic acid. Therefore, p-hydroxybenzoic acid is not a substance of very high concern (SVHC) according to Article 57, REACH.

 

4-HBA was selected as candidate CORAP substance according to article 44(1) REACH. The ground of concern was that 4-HBA is a suspected endocrine disruptor as published in the justification document on. However, a justification based on data is not given. Within the draft decision of ECHA / eMSCA (dated 19. August 2015 for the translation into German) an extended one-generation reproductive toxicity study in rats, oral route is requested to “… To clarify the concern on reproductive toxicity which could be possibly linked with endocrine disrupting modes (i.e. estrogenic activity) … ”

Again, a justification for the“concern”based on data is not given.

 

ECHA/ evaluating MSCA states:

“To clarify the concern on reproductive toxicity which could be possibly linked with endocrine disrupting modes (i.e. estrogenic activity) an extended one-generation reproduction toxicity study (B.46, OECD 443) is the preferred test.”

The registrants disagree with this statement.

With 4-HBA a GLP- and OECD 440 guideline-conform uterotrophic assay has been performed (Twomey 2000b). The uterotrophic bioassay according to EU B.54, OECD TG 440 in rodents is a short-term screening test for oestrogenic/ anti oestrogenic properties and is accepted by the ECHA guidance on information requirements (ECHA, 2015) as one of the available in vivo assays focusing on identification of endocrine disrupting potency. As this assay of Twomey (2000b) has been performed under an accepted quality assurance system using a guideline assay its results are fully reliable (Klimisch score 1). The assay has a negative result. Further, the uterotrophic assays with rats and mice published by Hossaini et al. (2000) are negative. These specific studies (uterotrophic assays) with repeated administration of dosages up to and including 100 mg/kg bw. for 3 consecutive days are available and are mainly used for assessment for this mode of action. The reliable uterotrophic assays (Twomey 2000b, Hossaini 2000) are negative for 4-hydroxybenzoic acid in rats and mice in concentrations up to and including 100 mg/kg bw. /day. Hence, it is concluded, that 4-hydroxybenzoic acid has no estrogenic activity. In 2013, the Scientific Committee on Consumer Safety (SCCS) published an updated scientific ‘Opinion’ (SCCS/1514/13). In this Opinion it was stated (p.6) that “…4-hydroxybenzoic acid (PHBA)…is considered to be an inactive metabolite” related to its endocrine disruptive properties. Therefore, we cannot agree with the statement of the ECHA/ evaluating MSCA: ”possible estrogenicity of the registered substance cause a concern…”. There exist three fully reliable studies with 4-HBA showing no endocrine disrupting effect. A detailed evaluation of the potential endocrine disruptive properties has been performed by the registrants and summarised in the updated Chemical Safety Report submitted to ECHA on March 31st 2015 already. It seems that this updated CSR with the evaluation of the potential endocrine disruptive properties had not been considered by the evaluating MSCA. Further, there exist studies with read-across substances methyl- and ethylparaben on fertility in rats revealing a lack of spermatotoxic effects (Oishi, 2004). In addition, within the repeated dose reproduction and developmental toxicity study of Nagao et al. (1997), reproductive organs in male and female rats (testes, epididymis, ovaries and uterus) as well as functional aspects on fertility (spermatogenesis, copulation index, fertility index, estrus cycles), which are assumed to be possibly affected by estrogens, were investigated. With these parameters no significant differences between 4-HBA treated animals and controls were observed. Hence, results of the reliable in vivo repeated dose-toxicity study in rats did not give any hint on an estrogenic activity of 4-hydroxybenzoic acid.

From these results and evaluations (in this section of the Chemical Safety Report as well as by SCCS) it can be concluded that there is no concern on reproductive toxicity which could be possibly linked with endocrine disrupting modes (i.e. estrogenic activity).

Thus, there is no rational basis for this draft decision of the ECHA/ evaluating MSCA requesting an extended one-generation reproduction toxicity study (B.46, OECD 443).

Furthermore, there exist 5 studies with mouse, rat, rabbit and hamster, on developmental toxicity with methylparaben and ethylparaben, precursors of 4-HBA (FDRL 1972, FDRL 1973, Moriyama et al. 1975). On a read-across basis (see attached document with the detailed read-across rationale, which is also part of the recently updated REACh registration dossier / Chemical Safety Report), these studies provide information required by Annex X, Section 8.7.2, REACH. No effects were observed.