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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Additional information

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.

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 impaired fertility after administration of p-hydroxybenzoic acid.

Data of methylparaben and ethylparaben can be taken for the evaluation of 4-HBA as there is a close structural similarity and the fact that 4-HBA is the main breakdown product of the precursor methylparaben and ethylparaben after oral administration.Justification for read-across: similar chemical structure (common functional group) and common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals (for justification see appendix to the Chemical Safety Report in IUCLID section 13).

A reliable experimental study on pre-natal developmental toxicity, equivalent to OECD 414, with administration of oral dosages of 0, 5.5, 25.5, 118, and 550 mg/kg bw of methylparaben to groups of 22-24 female rats (FDRL, 1972) is available which equals dosages of up to 499[1]mg/kg bw 4-HBA based on the knowledge about the hydrolysis of methylparaben into 4-HBA and methanol, the information of toxicokinetic experiments with methylparaben yielding 4-HBA and the molecular weight ratio of 4-HBA and methylparaben. Further 3 reliable studies equivalent to OECD 414 are available, with administration of similar oral dosages to mice, hamsters (FDRL, 1972) and rabbits (FDRL, 1973). In a non-guideline study, Moriyama et al. (1975) evaluated the developmental toxicity of ethylparaben at three dose levels (with a control group), with the highest dose level (estimated based on dietary intake) to be 2970-3260 mg/kg. Given the equimolar conversion of ethylparaben to 4-HBA, the 4-HBA equivalent exposure would be 2702 – 2967 mg/kg bw 4-HBA. In this study, fetal examinations included external and head examinations of all animals in each group, and soft tissue and skeletal examinations of half the animals in each group. For ethylparaben, the maternal and fetal NOAELs were set at 517 – 568 mg/kg based on maternal toxicity (decrease in food consumption) and visceral/skeletal abnormalities in offspring. Again, this would correspond to NOAELs of 470 – 517 mg/kg bw for 4-HBA equivalents. However, the observed effects are not specific for the test substance. The developmental effects were attributed to mothers malnutrition and a decrease in maternal food consumption appear to result from high amounts of test substance (10%) in the feed.

The current OECD Guideline for TG 414 (2001) also recommends that if a limit test of 1000 mg/kg does not produce effects, there may be no need for further testing.

Further, lack of spermatotoxic effects of methyl and ethyl esters of p-hydroxybenzoic acid was observed in rats (Oishi 2004, reviewed by BFR 2011). Based on available repeated-dose toxicity studies EFSA (2004) concluded that: “methyl and ethyl paraben showed no effects on sex hormones and the male reproductive organs in juvenile rats at dose levels up to 1000 mg/kg body weight/day. Therefore 1000 mg/kg body weight/day was considered a No Observed Adverse Effect Level (NOAEL) for both methyl paraben and ethyl paraben” (EFSA, 2004). The results of Oishi (2004) from read-across substances with lack of spermatotoxic effects can be attributed to 4-HBA as well.

With regard to the information requirement: Toxicity for reproduction / fertility (extended one-generation reproductive toxicity study) it is justified that no testing is necessary as follows:

Justification: A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) by oral gavage in male and female rats is reported for 4-hydroxybenzoic acid. The test was conducted with dose levels of 0, 40, 200, 1,000 mg/kg body weight/day for 42 days for males and 52 days for females starting from 2 weeks before mating to day 3 of lactation (Nagao et al. 1997).

In the cited study the animal number was not 20 but indeed only 13 and the pups were not divided 50/50 to be checked for bone or visceral malformations but 33/66. As only questionable toxicity was seen with the parenteral animals, we think the study to be acceptable and valid to evaluate the reproduction toxicity of 4-HBA.

A two-generation reproduction toxicity study or an extended one-generation reproductive toxicity study is not deemed necessary based on the following argumentation.

4-HBA is an organic acid. It is not irritating to the intact skin and is practically not resorbed through the skin. After oral dosages 4-HBA is resorbed relatively fast and it is relatively fast excreted with the urine. Relevant parts of the resorbed substance are excreted unchanged. It can be assumed that the excretion via kidneys is so fast that relevant parts of 4-HBA are excreted before they can be metabolized.

No accumulation was observed. One can assume that one reason for the missing toxic effect is the fast excretion from the body and this is also the reason why longer studies did not lead to or did not reveal toxic effects or stronger toxic effects. The toxic effects observed can be classed as unspecific changes caused by the acidic nature of 4-HBA. There are no indications on adverse effects on relevant reproductive parameters from available reproductive toxicity screening, subchronic and chronic repeated dose toxicity studies with 4-HBA and the read-across substances methyl- and ethylparaben.

Very high oral dosages impaired the acid-base-regulation and caused a metabolic acidosis in the blood. This explains the deviations in clinical chemistry and in haematology observed after high dosages of 4-HBA. In a 42 days repeated dose toxicity study with rats 40 mg/kg bw/day revealed to be the no-effect dosage. However, the changes at LOEL 200 mg/kg bw/day in clinical signs, haematology and clinical chemistry are not considered as adverse effects. NOAEL for systemic toxicity = 1,000 mg/kg bw/day. This evaluation is in line with the conclusion of the OECD SIDS (1999).

So the toxicity profile of 4-HBA can be fully derived from the existing studies and a read across from the toxicity studies with methyl- and ethyl-paraben also with respect to a possible reproduction toxicity and NO further testing such as an extended one-generation reproductive toxicity study is necessary. The reasons put forward above comply with Annex X of the REACH regulation in which it is stated:

When, for certain endpoints, it is proposed not to provide information for other reasons than those mentioned in column 2 of this Annex or in Annex XI, this fact and the reasons shall also be clearly stated.” (cited from Annex VII, VIII, IX, X, REACH). These reasons are clearly presented in this document (CSR).

For read across justification from the toxicity studies with methyl- and ethyl-paraben please refer to the attached document to the Chemical Safety Report (in section 13 of the IUCLID-Dossier).

.......................................

[1]100% hydrolysis is assumed



Short description of key information:
A screening reproductive/developmental toxicity study with rats revealed no effects on the parameters for fertility up to the highest dosage of 1000 mg/kg bw/day (Nagao et al., 1997).
NOEL = 1000 mg/kg bw/day
Further, lack of spermatotoxic effects of methyl and ethyl esters of p-hydroxybenzoic acid was observed in rats (Oishi 2004, reviewed by BFR 2011). Based on available repeated-dose toxicity studies EFSA (2004) concluded that: “methyl and ethyl paraben showed no effects on sex hormones and the male reproductive organs in juvenile rats at dose levels up to 1000 mg/kg body weight/day. Therefore 1000 mg/kg body weight/day was considered a No Observed Adverse Effect Level (NOAEL) for both methyl paraben and ethyl paraben” (EFSA, 2004). The results of Oishi (2004) from read-across substances with lack of spermatotoxic effects can be attributed to 4-HBA as well (for detailed read-across justification see appendix to the Chemical Safety Report in section 13 of IUCLID).

Effects on developmental toxicity

Description of key information
Please refer to the discussion section below, because the maximum size limit of this field is too little (2000 characters).
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Additional information

A screening reproductive/developmental toxicity study with rats revealed no effects on the developmental/ teratogenic parameters up to the highest dosage of 1000 mg p-hydroxybenzoic acid/kg bw. Key parameters of developmental toxicity such as external deformities andmorphological abnormalities in the autopsywere considered on lactation day 4.

While the study by Nagao et al. (1997) does not explicitly state that skeletal and visceral alterations were evaluated, the study report states that in the case of effects on the pups: “All cases were sacrificed on lactation day 4 and dissected. The organs in the thoracic and abdominal cavities were excised together and were fixed in 10% formalin and preserved according to litter. "Furthermore, the “pup findings” state that "No external deformities were observed in the surviving pups on the day of birth. In autopsies of pups on lactation day 4 as well, no abnormalities were observed in the control group, nor were any abnormalities observed in the autopsy of the dead pup. ”Taken together, these statements imply that the lack of details results from the absence of effects, rather than from the organs not being examined.

Developmental toxicity of p-hydroxybenzoic acid in rats was also investigated by Kavlock (1990). Single oral treatment on gestation day 11 up to the highest dosage of 1000 mg p-hydroxybenzoic acid/kg bw did not cause effects on developmental/ teratogenic parameters. No significant change in pup weight and overt malformation were observed. This is in line with results from Nagao et al. (1997) and supports the conclusion that p-hydroxybenzoic acid is not developmental toxic or teratogenic.

From the existing studies the toxicity of 4-HBA can be completely evaluated. Not only the toxic effects but also a rough idea of their dose-response relationship can be established. This is also true for the embryotoxic effects.

4-HBA is readily absorbed in the stomach and probably also from the respiratory tract. The resorption through the intact skin is very poor. The findings of 4-HBA in the urine can be explained by a relatively fast excretion in the kidney, although 4-HBA is also easily metabolised. The degradation is demonstrated in the environment but it should be also true for the metabolism in the human body.

Systemic toxic effects arise with oral dosages higher than 40 mg/kg bw or inhalative exposures greater than 60 mg/m³.

With the exception of the corrosive action of pure 4-HBA on mucous membranes (eye) the observed toxic effects are deviations in the range of the possible physiologic reaction of the body.

They can be explained by the pharmacologic action on the blood clotting time and by the disturbance of the acid-base equilibrium in the blood when higher dosages are administered.

Data of methylparaben and ethylparaben can be taken for the evaluation of 4-HBA as there is a close structural similarity and the fact that 4-HBA is the main breakdown product of the precursor methylparaben and ethylparaben after oral administration.Justification for read-across: similar chemical structure (common functional group) and common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals (for justification see appendix to the Chemical Safety Report in section 13 of IUCLID).

A reliable experimental study on pre-natal developmental toxicity, equivalent to OECD 414, with administration of oral dosages of 0, 5.5, 25.5, 118, and 550 mg/kg bw of methylparaben to groups of 22-24 female rats (FDRL, 1972) is available which equals dosages of up to 499[1]mg/kg bw 4-HBA based on the knowledge about the hydrolysis of methylparaben into 4-HBA and methanol, the information of toxicokinetic experiments with methylparaben yielding 4-HBA and the molecular weight ratio of 4-HBA and methylparaben. Further 3 reliable studies equivalent to OECD 414 are available, with administration of similar oral dosages to mice, hamsters (FDRL, 1972) and rabbits (FDRL, 1973). In a non-guideline study, Moriyama et al. (1975) evaluated the developmental toxicity of ethylparaben at three dose levels (with a control group), with the highest dose level (estimated based on dietary intake) to be 2970-3260 mg/kg. Given the equimolar conversion of ethylparaben to 4-HBA, the 4-HBA equivalent exposure would be 2702 – 2967 mg/kg bw 4-HBA. In this study, fetal examinations included external and head examinations of all animals in each group, and soft tissue and skeletal examinations of half the animals in each group. For ethylparaben, the maternal and fetal NOAELs were set at 517 – 568 mg/kg based on maternal toxicity (decrease in food consumption) and visceral/skeletal abnormalities in offspring. Again, this would correspond to NOAELs of 470 – 517 mg/kg bw for 4-HBA equivalents. However, the observed effects are not specific for the test substance. The developmental effects were attributed to mothers malnutrition and a decrease in maternal food consumption appear to result from high amounts of test substance (10%) in the feed.

The current OECD Guideline for TG 414 (2001) also recommends that if a limit test of 1000 mg/kg does not produce effects, there may be no need for further testing.

In the FDRL study with rats (1972), body weights were recorded as described in OECD 414 but missing 2 weight measurements during dosing period out of 7 measurements in total. Nevertheless, the average body weights increased continuously during dosing period in a dose-independent manner. A different picture is not expected to emerge by including two additional intermediate time points of weight measurement. As body weight is increasing over time, independent of dosage, no maternal effects are indicated. Likewise no key parameter on developmental toxicity appears to be missing for evaluation.

From these results, methylparaben and ethylparaben can be considered as not embryotoxic and not teratogenic.

Including the read across from data of methylparaben and ethylparaben the reproduction toxicity of 4-HBA can be evaluated completely. Therefore, further animal studies e. g. two generation/ extended one generation reproduction toxicity study or a study with 4-HBA according to OECD 414[2]will not give additional information or increase the safety. In accordance with the “rules for adaptation of the standard testing regime” according to Annex XI, REACH the existing data can be used in a “weight of evidence” (Annex XI (1.2) REACH) and a “read across approach” (Annex XI (1.5), REACH). The results of the studies cover all the following requested criteria for read across approach of REACH, Annex XI (1.5) as demonstrated above and in the robust study summaries of the technical IUCLID dossier:

Results are

  • “adequate for the purpose of classification and labelling and/or risk assessment,
  • have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3),
  • cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3) if exposure duration is a relevant parameter, and
  • adequate and reliable documentation of the applied method” were “provided.”

 

The results of the available studies (equivalent to OCED 414) on rats, mice and hamsters (FDRL, 1972) and rabbits (FDRL, 1973) for methylparaben, the study with rats (equivalent to OCED 414) with ethylparaben (Moriyama, 1975), the study with rats (eq. to OECD 422) with 4-HBA (Nagao, 1997) and the results on the toxicokinetics especially of the studies of Jones et. al (1956) with methylparaben, ethylparaben and 4-HBA which is also a key citation used in the OECD SIDS summary (OECD SIDS, 1999) and EFSA (2012) the substance evaluation for the endpoint developmental toxicity is considered as a “sufficient weight of evidence from several independent sources of information leading to the assumption/conclusion” that “the substance has” … “not a particular dangerous property” (fulfilling REACH, Annex XI, 1.2, first paragraph).

Thus, “sufficient weight of evidence for the” … “absence of a particular dangerous property is available” and:

  • “further testing on vertebrate animals for that property shall be omitted,
  • further testing not involving vertebrate animals may be omitted” (fulfilling the requirements in REACH, Annex XI, 1.2, third paragraph).

Adequate and reliable documentation is provided as appendix to the Chemical Safety Report attached in section 13 of the technical registration dossier (IUCLID) thus fulfilling the requirements in REACH, Annex XI, 1.2, last paragraph).


[1]100% hydrolysis is assumed

[2]Four reliable studies with rats, mice, hamsters and rabbits equivalent to OECD 414 with methylparaben are available. One reliable developmental toxicity study with ethylparaben is available.

Justification for classification or non-classification

4-HBA is not classified with respect to toxicity to reproduction according to the rules of regulation (EC) No. 1272/2008 because there were no observed adverse effects regarding fertility and developmental toxicity in available studies with 4-HBA and the source substances methylparaben and ethylparaben used in the read-across approach.