Predicting points of departure and potency categories for fragrance ingredients by integrating OECD in vitro models.

Continuous potency assessment is crucial for conducting quantitative risk assessment (QRA) of sensitizers. Quantitative regression models, based on in vitro methods, have been developed to calculate points of departure for use in skin sensitization QRA. These models calculate a point of departure as a predicted value for Local Lymph Node Assay (LLNA) EC3 or potency value (PV), integrating data from the kinetic Direct Peptide Reactivity Assay (kDPRA), KeratinoSens (KS) assay, and human Cell Line Activation Test (h-CLAT). The goal of this study was to determine how in vitro predicted EC3s and PVs compare to published reference data. In vitro data were combined in point of departure regression models to predict EC3s and PVs. These points of departure were then grouped into sensitization potency categories, such as extreme, strong, moderate, weak, very weak, or non-sensitizer, as previously described. Trends in potency distribution and high concordance between predicted EC3 and PV categories and published potency categories were observed. Furthermore, the median absolute fold-misprediction ranged between 1.8 and 2.5 for models predicting EC3 and between 1.7 and 3.4 for those predicting PVs. These regression models are a promising animal alternative for determining sensitization quantitative potency for fragrance ingredients, thereby facilitating QRA.

Quaternary ammoniums activate human dendritic cells and induce a specific T-cell response in vitro.

BACKGROUND: In many countries, neuro-muscular blocking agents (NMBAs) are the first cause of perioperative anaphylaxis. Epidemiological studies identified pholcodine, a quaternary ammonium-containing opiate as one of the sensitization sources. However, NMBA anaphylaxis exists in countries where pholcodine was unavailable, prompting the hypothesis of other sensitizing molecules, most likely quaternary ammonium compounds (QACs). Indeed, QACs are commonly used as disinfectants, antiseptics, preservatives, and detergents. Occupational exposure to QACs has been reported as a risk factor for NMBA anaphylaxis, but little is known about the sensitization mechanism and the capacity of these molecules to elicit an immune response. We aimed to establish the immunogenicity of QACs representative of the main existing chemical structures. METHODS: We measured the sensitization potential of seven QACs (two polyquaterniums, three alkyl-ammoniums and two aromatic ammoniums) by using two standard dendritic cells (DCs) models (THP-1 cell line and monocyte derived-dendritic cells). The allergenicity of the sensitizing compounds was further tested in heterologous and autologous T-cell-DC co-culture models. RESULTS: Amongst the seven molecules tested, four could modulate activation markers on DCs, and thus can be classified as chemical sensitizers (polyquaterniums-7 and -10, ethylhexadecyldimethylammonium and benzethonium). This activation was accompanied by the secretion of pro-inflammatory and maturation cytokines. Furthermore, activation by polyquaternium-7 could induce T-cell proliferation in heterologous and autologous coculture models, demonstrating that this molecule can induce a specific CD4+ T cell response. CONCLUSIONS: We provide evidence at the cellular level that some QACs can elicit an immune response, which could be in line with the hypothesis of these molecules' role in NMBA sensitization.

In vitro to in vivo extrapolation to derive a metabolism factor for estimating the aggregate exposure to salicylic acid after dermal exposure of its esters.

As part of the safety assessment of salicylate esters in cosmetics, we developed a metabolism factor based on in vitro to in vivo extrapolation (IVIVE) to provide a better estimation of the aggregate internal exposure to the common metabolite, salicylic acid. Optimal incubation conditions using human liver S9 were identified before measuring salicylic acid formation from 31 substances. Four control substances, not defined as salicylic esters but which could be mistaken as such due to their nomenclature, did not form salicylic acid. For the remaining substances, higher in vitro intrinsic clearance (CLint, in vitro) values generally correlated with lower LogP values. A "High-Throughput Pharmacokinetic" (HTPK) model was used to extrapolate CLint, in vitro values to human in vivo clearance and half-lives. The latter were used to calculate the percentage of substance metabolised to salicylic acid in 24 h in vivo following human exposure to the ester, i.e. the "metabolism factor". The IVIVE model correctly reproduced the observed elimination rate of 3 substances using in silico or in vitro input parameters. For other substances, in silico only-based predictions generally resulted in lower metabolism factors than when in vitro values for plasma binding and liver S9 CLint, in vitro were used. Therefore, in vitro data input provides the more conservative metabolism factors compared to those derived using on in silico input. In conclusion, these results indicate that not all substances contribute equally (or at all) to the systemic exposure to salicylic acid. Therefore, we propose a realistic metabolism correction factor by which the potential contribution of salicylate esters to the aggregate consumer exposure to salicylic acid from cosmetic use can be estimated.

Avoiding a reproducibility crisis in regulatory toxicology-on the fundamental role of ring trials.

The ongoing transition from chemical hazard and risk assessment based on animal studies to assessment relying mostly on non-animal data, requires a multitude of novel experimental methods, and this means that guidance on the validation and standardisation of test methods intended for international applicability and acceptance, needs to be updated. These so-called new approach methodologies (NAMs) must be applicable to the chemical regulatory domain and provide reliable data which are relevant to hazard and risk assessment. Confidence in and use of NAMs will depend on their reliability and relevance, and both are thoroughly assessed by validation. Validation is, however, a time- and resource-demanding process. As updates on validation guidance are conducted, the valuable components must be kept: Reliable data are and will remain fundamental. In 2016, the scientific community was made aware of the general crisis in scientific reproducibility-validated methods must not fall into this. In this commentary, we emphasize the central importance of ring trials in the validation of experimental methods. Ring trials are sometimes considered to be a major hold-up with little value added to the validation. Here, we clarify that ring trials are indispensable to demonstrate the robustness and reproducibility of a new method. Further, that methods do fail in method transfer and ring trials due to different stumbling blocks, but these provide learnings to ensure the robustness of new methods. At the same time, we identify what it would take to perform ring trials more efficiently, and how ring trials fit into the much-needed update to the guidance on the validation of NAMs.

The trilogy of human musk receptors: linking receptor activation, genotype, and sensory perception.

The scent of musk plays a unique role in the history of perfumery. Musk odorants comprise 6 diverse chemical classes and perception differences in strength and quality among human panelists have long puzzled the field of olfaction research. Three odorant receptors (OR) had recently been described for musk odorants: OR5AN1, OR1N2, and OR5A2. High functional expression of the difficult-to-express human OR5A2 was achieved by a modification of the C-terminal domain and the link between sensory perception and receptor activation for the trilogy of these receptors and their key genetic variants was investigated: All 3 receptors detect only musky smelling compounds among 440 commercial fragrance compounds. OR5A2 is the key receptor for the classes of polycyclic and linear musks and for most macrocylic lactones. A single P172L substitution reduces the sensitivity of OR5A2 by around 50-fold. In parallel, human panelists homozygous for this mutation have around 40-60-fold higher sensory detection threshold for selective OR5A2 ligands. For macrocyclic lactones, OR5A2 could further be proven as the key OR by a strong correlation between in vitro activation and the sensory detection threshold in vivo. OR5AN1 is the dominant receptor for the perception of macrocyclic ketones such as muscone and some nitromusks, as panelists with a mutant OR5A2 are still equally sensitive to these ligands. Finally, OR1N2 appears to be an additional receptor involved in the perception of the natural (E)-ambrettolide. This study for the first time links OR activation to sensory perception and genetic polymorphisms for this unique class of odorants.

Bioconcentration Assessment in Fish Based on In Vitro Intrinsic Clearance: Predictivity of an Empirical Model Compared to In Vitro-In Vivo Extrapolation Models.

To estimate the bioconcentration factor (BCF), the in vitro intrinsic clearance (CLIN VITRO,INT) from rainbow trout liver S9 fractions (RT-S9) can be applied to in vitro-in vivo extrapolation (IVIVE) models, yet uncertainties remain in model parameterization. An alternative model approach is evaluated: a regression model was built in the form log BCF = a × log Kow + b × log CLIN VITRO,INT. The coefficients a and b were fitted based on a training set of 40 chemicals. A high robustness of the coefficients and good accuracy of BCF prediction were found on independent datasets of neutral organic chemicals (measured log Kow 3.3-6.2). BCF predictions were similar to or in better agreement with in vivo BCFs compared to IVIVE models (2.4- to 2.9- vs 2.8- to 3.6-fold misprediction) for training and test sets. Species-matched models (trout, carp) did not result in improvements. This study presents the largest dataset on CLIN VITRO,INT and BCFs to assess predictivity of the RT-S9 assay. The robustness of the regression statistics on different datasets and the high statistical weight of the CLIN VITRO,INT term illustrate the predictive power of the RT-S9 assay as an important step toward regulatory acceptance to replace animal experiments.

Integrated skin sensitization assessment based on OECD methods (III): Adding human data to the assessment.

Skin sensitizer potency assessment based on new approach methodologies is key to deriving a point of departure (PoD) for risk assessment. Regression models to predict a PoD based on OECD-validated in vitro tests and trained on local lymph node assay (LLNA) data were previously presented, and results from human tests were recently compiled. To integrate both data sources, the Reference Chemical Potency List (RCPL), which provides potency values (PV) for 33 chemicals integrating LLNA and human data in a structured weight-of-evidence approach, was developed. When calculating regression models vs PV or LLNA data, different weights for the input parameters were noted. As the RCPL is based on too few chemicals to train robust statistical models, the list of human data was extended to a larger set of PV (n = 139) with associated in vitro data. This database was used to retrain the regression models and to compare regression models trained vs (i) LLNA, (ii) PV or (iii) human DSA04 values. Using the PV as a target, predictive models of similar predictivity to the LLNA-based models were obtained, which mainly differ in a lesser weight of cytotoxicity and a higher weight of cell activation and reactivity parameters. Analysis of the human DSA04 dataset indicates a similar pattern but also shows that the human dataset is too small and biased to be a key dataset for potency prediction. Hence, an enlarged set of PV values appears to be a complementary tool to train predictive models next to an LLNA-only database.

ECHA ARN documents: chemical grouping without a toxicological rationale.

The EU chemical strategy for sustainability (CSS) plans to use chemical grouping to "prioritise (…) substances for restrictions for all uses through grouping, instead of regulating them one by one". Thus, toxicological grouping will become a key tool used by regulatory authorities in Europe. Over the last 2 years, ECHA has published a high number of documents labelled "Assessment of Regulatory Needs (ARN)" which are based on groups of chemicals based on structural considerations. The ARN documents are legally non-binding, yet they present the public impression of a conclusion about restrictions for groups or sub-groups of chemicals and hence may set a precedent for further binding actions. ECHA has set out definitions on what is considered a group in REACH Annex XI. However, as shown in this commentary based on five examples, the ARN do not follow these principles and propose toxicological groupings without taking into consideration mode of action and the toxicological information on the chemicals. Given the emphasis on grouping projected by the CSS, the groupings in the ARN set an unfortunate precedent on what a toxicological group means and they do not follow clear scientific standards or established toxicological principles. They also lead to a public image of guilt by association for chemicals, without any recourse for registrants to establish the scientific basis for their safe use, as presented within REACH registrations.

Reduced specificity for the local lymph node assay for lipophilic chemicals: Implications for the validation of new approach methods for skin sensitization.

Meaningful and accurate reference data are crucial for the validation of New Approach Methodologies (NAMs) in toxicology. For skin sensitization, multiple reference datasets are available including human patch test data, guinea pig data and data from the mouse local lymph node assay (LLNA). When assessed against the LLNA, a reduced sensitivity has been reported for in vitro and in chemico assays for lipophilic chemicals with a LogP ≥3.5, resulting in reliability restrictions within the h-CLAT OECD test guideline. Here we address the question of whether LLNA data are an appropriate reference for chemicals in this physicochemical range. Analysis of LLNA vs human reference data indicates that the false-discovery rate of the LLNA is significantly higher for chemicals with LogP ≥3.5. We present a mechanistic hypothesis whereby irritation caused by testing lipophilic chemicals at high test doses leads to unspecific cell proliferation. The accompanying analysis indicates that for lipophilic chemicals with negative calls in in vitro and in chemico assays, resorting to the LLNA is not necessarily a better option. These results indicate that the validation of NAMs in this particular LogP range should be based on a more holistic evaluation of the reference data and not solely upon LLNA data.

Towards the mechanism of spermatotoxicity of p-tert-butyl-alpha-methylhydrocinnamic aldehyde: inhibition of late stage ex-vivo spermatogenesis in rat seminiferous tubule cultures by para-tert-butyl- benzoic acid.

Molecules metabolized to para-tert-butyl-benzoic acid (p-TBBA) affect male reproduction in rats through effects on spermatogenesis. This toxicity is specific to p-TBBA and not observed in meta-substituted analogues. The underlying mode of action was evaluated by comparing effects of p-TBBA and the position isomer m-TBBA (2-50 µM) in an ex vivo 3D primary seminiferous tubule cell culture system from juvenile Sprague Dawley rats (Bio-AlteR®). Treated cultures were evaluated for CoA-conjugate formation, cytotoxicity, blood-testis barrier functionality and different germ cell populations to assess effects on spermatogenesis. In addition, an evaluation of the metabolome of treated cultures was performed by using MxP® Broad Profiling via a LC-MS/MS and GC-MS platform. Para-TBBA decreased germ cell populations of late stages of spermatogenesis and led to the formation of CoA-conjugates in the ex vivo tissue. In addition, p-TBBA had a pronounced effect on the metabolome by affecting lipid balance and other CoA-dependent pathways contributing to energy production and the redox system. Meta-TBBA did not affect germ cell populations and no m-TBBA related CoA-conjugates were detectable. The metabolic profile of m-TBBA treated cells was comparable to vehicle control treated cultures, indicating that formation of CoA-conjugates, inhibition of spermatogenesis, and effects on the metabolome are mechanistically linked events. Thus, for this specific chemical group an adverse outcome pathway can be postulated, including the formation of benzoic acid metabolites, accumulation of CoA-conjugates to a certain threshold and CoA depletion, which affects the metabolic and lipid profile and leads to tissue specific effects with impaired functionalities such as spermatogenesis.

Reference Chemical Potency List (RCPL): A new tool for evaluating the accuracy of skin sensitisation potency measurements by New Approach Methodologies (NAMs).

Considerable progress has been made in the design of New Approach Methodologies (NAMs) for the hazard identification of skin sensitising chemicals. However, effective risk assessment requires accurate measurement of sensitising potency, and this has proven more difficult to achieve without recourse to animal tests. One important requirement for the development and adoption of novel approaches for this purpose is the availability of reliable databases for determining the accuracy with which sensitising potency can be predicted. Some previous approaches have relied on comparisons with potency estimates based on either human or animal (local lymph node assay) data. In contrast, we here describe the development of a carefully curated Reference Chemical Potency List (RCPL) which is based on consideration of the best available human and animal data. The RCPL is comprised of 33 readily available chemicals that span a wide range of chemistry and sensitising potency, and contain examples of both direct and indirect (pre- and pro-) haptens. For each chemical a potency value (PV) was derived, and chemicals ranked according to PV without the use of potency categories. It is proposed that the RCPL provides an effective resource for assessment of the accuracy with which NAMs can measure skin sensitising potency.

Nature-derived epoxy resins: Synthesis, allergenicity, and thermosetting properties of pinoresinol diglycidyl ether.

We describe a novel nature-derived epoxy resin monomer (ERM) derived from the plant lignan pinoresinol. Epoxy resins are thermosetting materials in global usage owing to their excellent technical properties such as flexibility and durability. However, their adverse health effects are often not considered and affect users of epoxy resins worldwide. Components of epoxy resin systems are strong skin sensitizers and cause allergic contact dermatitis. The reported prevalence attributable to epoxy chemicals is between 11.7 and 12.5% of all cases of occupational allergic contact dermatitis. We are committed to developing epoxy resins with reduced allergenic effect, while maintaining their excellent properties. The novel ERM, pinoresinol diglycidyl ether (PinoDGE), was synthesized in one step from pinoresinol and epichlorohydrin in 88% yield. It was not classified as a skin sensitizer in the in vivo local lymph node assay, at concentrations up to 0.17 m, as it did not cause a stimulation index >3 compared to control. Pinoresinol diglycidyl ether reacted with the model peptide AcPHCKRM in a reactivity assay and was predicted to be a skin sensitizer in the KeratinoSens assay. Preliminary cross-linking studies indicate that it has promising properties compared to commercially used ERMs. Pinoresinol diglycidyl ether could be seen as a lead compound for further development of alternative ERMs with a better safety profile based on natural and renewable sources for construction of epoxy resin polymers.

Integrated skin sensitization assessment based on OECD methods (II): Hazard and potency by combining kinetic peptide reactivity and the "2 out of 3" Defined Approach.

Depending on regulatory requirements, the skin sensitization risk for new chemicals with potential consumer skin contact must be assessed by experimental testing by (i) binary hazard assessment to identify sensitizers, (ii) subclassification of sensitizers according to the Global Harmonized System (GHS), and (iii) derivation of a point of departure (PoD) for risk assessment. The Organisation for Economic Co-operation and Development (OECD) recently published a test guideline incorporating the "2 out of 3" defined approach (2o3 DA) for skin sensitization hazard assessment and added the kinetic direct peptide reactivity assay (kDPRA) as a stand-alone test guideline method for GHS subclassification. The 2o3 DA requires that at least two in vitro tests are conducted. The cell-based tests and the kDPRA generate, next to a binary outcome with a fixed threshold, continuous concentration-response data, which can be used in quantitative regression models to derive a PoD. The sequence of testing for the 2o3 DA is flexible. Here we compare different testing sequences and how they can be combined with kDPRA data to provide a PoD in parallel to hazard identification (hazard ID) and GHS subclassification. A set of 188 chemicals with available in vitro data was evaluated for the final PoD using these dif-ferent testing sequences. The results indicate that testing can start with DPRA / kDPRA and either of the cell-based assays, and that testing can stop after two congruent tests without major impact on the final PoD for chemicals within the applica-bility domain of the kDPRA.

Integrated skin sensitization assessment based on OECD methods (I): Deriving a point of departure for risk assessment.

Three guidelines covering key events in the skin sensitization adverse outcome pathway are endorsed by the Organisation for Economic Co-operation and Development (OECD). A recent guideline covers defined approaches (DA) to combine data from these tests for regulatory (sub)classification. The guideline methods provide continuous data that could charac-terize the sensitization potency on a more granular scale beyond (sub)classifications. We assembled a comprehensive database of in vitro and in vivo results from OECD guideline tests. Building on a previous approach using regression models, we provide quantitative models using input data from the kinetic direct peptide reactivity assay (kDPRA), the KeratinoSens™ (KS) assay, and the human cell line activation test (h-CLAT) to calculate a point of departure (PoD) in the form of a predicted local lymph node assay (LLNA) EC3 value for use in risk assessment. Predictive models include results from either two or all three assays. Detailed analysis versus in vivo data estimates redundancy between different tests and helps guide model selection. All models were tested on a set of case studies selected for their availability of multiple LLNA reference data in the OECD database. The predicted PoDs were within or close to the range of the variation of the historical LLNA data for most of these cases studies, and overall, the models predicted the in vivo value with a median fold-misprediction factor of around 2.5. The robustness of the models was characterized by comparing a comprehensive historical database versus the curated dataset provided by the OECD working group on DA.

Scientific discrepancies in European regulatory proposals on endocrine disruptors-REACH regulation quo vadis?

The EU chemical strategy for sustainability places a high focus on endocrine-disrupting chemicals (ED), the importance of their identification with increased testing and a ban in consumer products by a generic approach. It is assumed that for ED no threshold and hence no safe dose exists, leading to this generic approach. This view appears to be linked to the claim that for ED 'low-dose non-monotonic dose response' (low-dose NMDR) effects are observed. Without this hypothesis, there are no scientific reasons why classical risk assessment cannot be applied to the ED mode-of-action. Thus, whether for ED low-dose NMDR effects are considered a reproducible scientific fact by European authorities is Gretchen's question in this politicized field. Recent documents by the SCCS, EFSA and ECHA reviewed herein illustrate the diverging views within European scientific bodies on this issue. Furthermore, ED researchers never replicated findings on low-dose NMDR in blinded inter-laboratory experiments and the CLARITY-BPA core studies could not find evidence for reproducible NMDR for BPA. ECHA proposes a battery of in vitro tests to test all chemicals for ED properties. However, these tests were never validated for relevance and their high positivity rate could lead to increased follow-up animal testing. Based on (i) lack of reproducibility data for low-dose NMDR, (ii) diverging views within European authorities on NMDR and (iii) lack of fully validated in vitro test methods it might be premature to fast-track the wide-ranging changes in the regulatory landscape proposed by the authorities ultimately leading to drastically increased animal testing.

A critical assessment of the estrogenic potency of benzyl salicylate.

Benzyl salicylate (BS) is a natural ingredient of essential oils and a widely used fragrance chemical. A number of in vitro screening studies have evaluated the estrogenic potential of BS with ambiguous results. Lack of dose-response information for the positive control 17ß-estradiol (E2) in most studies makes an assessment of the relative potency and efficacy challenging. Notwithstanding this difficulty, BS has been added as the only fragrance ingredient to the list of the first 14 substances to be screened as potential endocrine disruptors by the European Scientific Committee for Consumer Safety (SCCS) and it is included in the Community rolling action plan (CoRAP) of the European REACH regulation to be assessed for the same property. Here we review all literature evidence and present new data to quantify the in vitro potency and efficacy of BS vs. E2 with full dose response analysis in both an estrogen response element (ERE) depending reporter gene assay and in the MCF7 cell proliferation (E-screen) assay. In both assays, very similar results for BS were found. BS is a partial agonist exhibiting 35-47 % maximal efficacy and it is active only close to the cytotoxic concentration. The extrapolated concentration to achieve 50 % efficacy is 21'000'000 higher as compared to E2 in the reporter gene assay. A ca. 36'000'000 higher concentration of BS as compared to E2 is required to reach equivalent partial cell proliferation stimulation in the MCF7 proliferation assay. This potency is significantly below the agonistic activity of known chemicals which cause estrogenic effects in in vivo assays. Importantly, in this study the weak agonistic activity is for the first time directly related to the activity of E2 in a full quantitative comparison in human cell lines which may help ongoing evaluations of BS by regulatory bodies.

A triangular approach for the validation of new approach methods for skin sensitization.

The availability of reference data is a key requirement for the development of new approach methods (NAM), i.e., in vitro, in chemico and in silico methods and integrated approaches, like defined approaches (DA), which combine these data sources. Reference data are of even greater importance for regulatory acceptance. In contrast to most other adverse effects, human skin sensitization data on many chemicals are available, next to data from animal studies, such as the local lymph node assay (LLNA). Skin sensitization NAM data can therefore be compared to different reference datasets. Recent publications and validation at the OECD focused on human and LLNA reference data. The "2 out of 3" DA (2o3 DA) is the first DA for skin sensitization solely based on experimental data from validated tests and was recently adopted as an OECD test guideline. Here we review the predictivity of the 2o3 DA on multiple human and LLNA reference datasets. Concomitantly, we compare the predictivity of the LLNA for human data within the same datasets. Comparing predictivity of methods not only bilaterally (NAM or DA vs. animal method) but including human data in a triangle "NAM data ­ animal data ­ human data" offers a comprehensive assessment of the NAM's and DA's predictivity. In all these assessments, the 2o3 DA was superior to the LLNA in predicting human skin sensitization hazard. This highlights the importance of a holistic view of reference data instead of limiting validation of NAMs and DAs to data from a single animal test only.

A species specific metabolism leading to male rat reprotoxicity of Cyclamen aldehyde: in vivo and in vitro evaluation.

Cyclamen aldehyde (CA; 3-(4-isopropylphenyl)-2-methylpropanal) is a widely used fragrance material. Repeated dose studies in rats revealed adverse effects on sperm maturation. Here we review all the mechanistic and in vivo evidence, to determine relevancy to human health. The effect on spermatogenesis appears to be linked to the metabolite p-isopropyl-benzoic acid (p-iPBA). Studies in rat, rabbit and human suspended hepatocytes indicated species differences with p-iPBA detected in rat hepatocytes only. In plated rat hepatocytes, p-iPBA is conjugated to Coenzyme A (CoA) and p-iPBA-CoA accumulates to stable levels over 22 h. In vitro accumulation of CoA conjugates is a metabolic hallmark correlated to male rat reproductive toxicity for related compounds. p-iPBA-CoA is formed in vivo in liver and testes of rats dosed with CA. In plated rabbit and human hepatocytes p-iPBA-CoA doesn't accumulate. Correlating to this lack of metabolite accumulation, no effects of CA on spermatogenesis were observed in a rabbit in vivo study. A species specific metabolic fate linked to CA toxicity in male rats is postulated which appears not relevant to the rabbit as non-responder species. Lack of accumulation of p-iPBA-CoA in human hepatocytes indicates that like rabbits, humans are unlikely to be vulnerable to p-iPBA hepatic and testicular toxicity.

Benzoyl-CoA conjugate accumulation as an initiating event for male reprotoxic effects in the rat? Structure-activity analysis, species specificity, and in vivo relevance.

A number of para-substituted benzoic acids (p-BA) and chemicals metabolized to p-BA have been found to confer adverse effects in male rats on sperm viability, motility, and morphology. These effects are putatively associated with the metabolism of p-BA to toxic intermediates. We had shown that p-BA lead to accumulation of high levels of p-alkyl-benzoyl-CoA conjugates in plated primary rat hepatocytes. Here we further investigated the relevance of this metabolic pathway for the reprotoxic effects in rats and rabbits. We extended the structure-activity relationship to a set of 19 chemicals (nine reprotoxic and ten non-reprotoxic) and confirmed a very strong correlation between p-alkyl-benzoyl-CoA accumulation in rat hepatocytes and the toxic outcome. Species specificity was probed by comparing rat, rabbit and human hepatocytes, and p-benzoyl-CoA accumulation was found to be specific to the rat hepatocytes, not occurring in human hepatocytes. There was also very limited accumulation in hepatocytes from rabbits that are a non-responder species in in vivo studies. Tissues of rats treated with 3-(4-isopropylphenyl)-2-methylpropanal were analysed and p-isopropyl-benzoyl-CoA conjugates were detected in the liver and in the testes in animals at toxic doses indicating that the metabolism observed in vitro is relevant to the in vivo situation and the critical metabolite does also occur in the reproductive tissue. These multiple lines of evidence further support benzoyl-CoA accumulation as a key initiating event for a specific group of male reproductive toxicants, and indicate a species-specific effect in the rat.