Acute toxicity testing of chemicals—Opportunities to avoid redundant testing and use alternative approaches

Assessment of the acute systemic oral, dermal, and inhalation toxicities, skin and eye irritancy, and skin sensitisation potential of chemicals is required under regulatory schemes worldwide. In vivo studies conducted to assess these endpoints can sometimes be associated with substantial adverse effects in the test animals, and their use should always be scientifically justified. It has been argued that while information obtained from such acute tests provides data needed to meet classification and labelling regulations, it is of limited value for hazard and risk assessments. Inconsistent application of in vitro replacements, protocol requirements across regions, and bridging principles also contribute to unnecessary and redundant animal testing. Assessment of data from acute oral and dermal toxicity testing demonstrates that acute dermal testing rarely provides value for hazard assessment purposes when an acute oral study has been conducted. Options to waive requirements for acute oral and inhalation toxicity testing should be employed to avoid unnecessary in vivo studies. In vitro irritation models should receive wider adoption and be used to meet regulatory needs. Global requirements for sensitisation testing need continued harmonisation for both substance and mixture assessments. This paper highlights where alternative approaches or elimination of tests can reduce and refine animal use for acute toxicity requirements.     

Reducing the number of fish in bioconcentration studies with general chemicals by reducing the number of test concentrations

Fish bioconcentration test guidelines generally require that bioconcentration factors (BCFs) are determined at two exposure concentrations. However, recent revisions to the OECD test guideline for bioconcentration testing (TG 305) provide the option to use only one exposure concentration, when justification is provided, although two concentrations may still be required for some regulatory purposes. Recently, this justification has been demonstrated for plant protection product active ingredients. To determine whether this justification has a broader validity for general chemicals, an analysis of 236 BCF studies on general chemicals was conducted. The results presented here again demonstrate that BCF values do not significantly differ between concentrations when more than one concentration is used. This relationship is particularly strong for BCFs ?1000 L/kg, which is beneficial, since only chemicals with BCFs >2000 L/kg may require regulatory action. This analysis therefore provides a data-driven rationale for using the one test concentration approach for general chemical substances and thus could contribute to a substantial reduction in the use of fish in bioconcentration tests.     

Acute and chronic sensitivity,avoidance behavior and sensitive life stages of bullfrog tadpoles exposed to the biopesticide abamectin

As compared to other aquatic organism groups, relatively few studies have been conducted so far evaluating the toxicity of pesticides to amphibians. This may at least partly be due to the fact that regulations for registering pesticides usually do not require testing amphibians. The sensitivity of amphibians is generally considered to be covered by that based on toxicity tests with other aquatic organisms (e.g. fish) although the impact of a pesticide on amphibians may be very different. In the present study, acute and chronic laboratory tests were conducted to evaluate the acute and chronic toxicity of abamectin (as Vertimec^® 18EC) to bullfrog (Lithobates catesbeianus) tadpoles. Acute tests were conducted at two tadpole stages (Gosner stage 21G and 25G) and avoidance tests were also conducted with stage Gosner stage 21G tadpoles. Calculated acute toxicity values were greater than those reported for standard fish test species, hence supporting the use of fish toxicity data as surrogates for amphibians in acute risk assessments. Given the limited number and extent of available amphibian toxicity studies, however, research needs to increase our understanding of pesticide toxicity to amphibians are discussed.     

Optimising the design of preliminary toxicity studies for pharmaceutical safety testing in the dog

A working party, comprising two animal welfare organisations and some 12 pharmaceutical companies in Europe, was established to minimise the use of the dog in safety testing. As first step, the participants defined the major objectives of preliminary dose-range finding/MTD toxicity studies in non-rodents, defined the principles and requirements for this study type and agreed on a proposal for an optimised study design, based on collective experience of conducting such studies in industry, involving an evaluation of 100 individual study data sets. The suggested study design is explained and described, and reflects current best practice in the pharmaceutical industry in Europe. The implementation of such an optimised design is believed to result in a reduction in the overall numbers of animals used for this purpose, without jeopardising the scientific rationale and usefulness of the studies for informing the conduct of later regulatory studies.     

ED points and NOELs: how they are used by UK pesticide regulators

The availability and use of ED point and NOEL values in ecotoxicological data submitted to the UK Pesticides Safety Directorate is explored. In first tier data normally submitted for pesticide registration, ED points such as EC/LC/LD50s are commonly available from acute laboratory toxicity studies conducted using birds, small mammals, fish, aquatic invertebrates, plants, honeybees and earthworms. These ED points are subsequently used for hazard classification of the pesticide and in toxicity: exposure ratio (TER) calculations required during the acute risk assessment. Although NOELs can often be available from the same first tier data, they are generally not used. However, NOELs commonly available from higher tier chronic toxicity studies such as chronic fish, Daphnia reproduction and avian reproduction studies, are used in TER calculations to assess chronic risk. The statistical limitations of the NOEL are recognized and the regulatory implications of replacing the NOEL with an alternative ED point are discussed.     

Aquatic test organisms and methods useful for assessment of chronic toxicity of chemicals

Literature data on the toxicity of chemicals to fish, a daphnid, and a mammal were reviewed to form a basis for efficiently determining chronic no-effect levels for many chemicals as may be required by law. The principal measurements used in statistically determining chronic no-effect levels for chemicals are the acute LC₅₀, the maximum acceptable toxicant concentration (MATC), and the application factor (AF). Equations for these measurements were developed to help in predictive calculation of AFs, MATCs and LC₅₀'s for combinations of related and unrelated organisms (fish and Daphnia) and chemicals. None of these methods are entirely reliable but are useful for early assessment of chronic toxicity to determine whether extensive testing appears necessary. The degree of predictability increases with the use of shortened or short-life-cycle chronic toxicity data.     

Harmonization of guidelines for toxicity testing of pharmaceuticals by 1992

In the past there has been considerable disagreement between various regulatory authorities regarding the type and design of animal tests that should be required before a new medicine can be used ethically and safely in the clinic. However, regulatory variations have largely been removed within politically and geographically similar regions (e.g., the U.S.A., the European Community, the Nordic countries) and there now appears to be a consensus regarding the value of harmonizing international requirements. In order to assist the process of harmonization, a detailed table of preclinical toxicity requirements in the U.S.A., Canada, Japan, and the European Community for each test (acute, subacute, chronic, carcinogenicity, mutagenicity, reproduction) has been compiled. This has been circulated to the relevant regulatory authorities to ensure that it accurately reflects current requirements. The major differences between authorities were found to be the duration of chronic, repeated-dose tests and the design of reproduction studies. International pharmaceutical companies were asked to complete a questionnaire, indicating how they design their preclinical testing program to comply with varying regulatory requirements. Most of the respondent companies indicated that chronic tests of longer than 6 months were conducted solely to comply with some regulatory requirements. Many companies repeat reproduction studies in order to comply with Japanese requirements. This emphasizes the need to harmonize these guidelines and discussions are currently underway to attempt to develop protocols acceptable to the FDA, the EC, and the Japanese Ministry of Health and Welfare.     

A strategy to reduce the use of fish in acute ecotoxicity testing of new chemical substances notified in the European Union

This study explores the applicability of a fish acute threshold (step-down) test approach for the assessment of new chemical substances notified in the EU. The proposed approach basically implies replacing the fish LC50 toxicity test with a simple acute threshold test and thus reducing the number of fish used and also costs. The fish test would be performed only at one concentration, the lowest between the EC50 concentrations obtained with previous testing with algae and daphnia. When fish would be more sensitive than algae and daphnia, testing with fish would be continued at lower concentrations (step-down). From step-down test results the LC50 value can be obtained by applying the binominal method of interpolation. These data can be used together with algal and daphnid data to provide the same Predicted No Effect Concentration values. The acute aquatic toxicity data used in this evaluation were extracted from the New Chemicals Database of the European Chemicals Bureau. The results show that 53.6-71.2% reduction of the number of fish used would be possible when applying this new testing strategy and suggest its use for regulatory purposes.     

Ambient bioassays for assessing water-quality conditions in receiving streams

The purpose of this paper is to show how short-term laboratory tests, conducted according to US Environmental Protection Agency (EPA) procedures, can be used effectively to assess water-quality conditions in streams or rivers that receive pollutants from industry or municipal or agricultural areas. Standardized, short-term tests with fish, aquatic invertebrates or algae are commonly used to estimate the acute or chronic toxicity of wastewaters; this is referred to as effuent testing. The methods used for testing effluents also can be used to assess water quality in receiving streams: in this application, the procedures are referred to as ambient testing. Despite similarity in methods, the major objective of effluent testing differs importantly from that of ambient testing. In effluent testing, the key objective is to determine how toxic an effluent is; in ambient testing, the main objective usually is that of determining if the water at a site is toxic. This difference is subtle but very important: it shapes the strategy for cost-effective ambient testing, and determines the framework for effective statistical analysis and interpretation of ambient toxicity test results. Specific case-study examples are provided demonstrating the kinds of information that can be extracted from ambient toxicity testing by use of different statistical methods, including analysis of variance, contingency-table analysis, and two types of multivariate procedures (principal components analysis and logistic regression). Examples also are given supporting the idea that an effective ambient testing programme should be long-term, and contain a diagnostic-testing component analogous to the toxicity identification procedures used to supplement effluent-testing programmes. Recommendations derived as lessons learned from largescale ambient toxicity testing programmes for receiving streams at Department of Energy facilities include: (1) testing more frequently with one species (preferably Ceriodaphnia) generally is more effective, in terms of information gained per dollar spent, than testing less frequently with two or more species; (2) use five or more sites per test period, plus two or more reference sites, whenever possible; (3) use four to six test periods per year; and (4) use diagnostic testing to supplement the ambient-testing programme. Various laboratory and in situ methods for environmental assessment are now under development, but these methods probably will not gain acceptance for use in regulatory situations for many years. Rapid growth in need for ecological risk assessments outstrips the rate at which new test procedures are approved for regulatory purposes. Thus, laboratory tests for estimating possible environmental impacts of toxic or disruptive pollutants are likely to be used more frequently during the next decade.     

Comparative analyses of cellular physiological responses of non-target species to cypermethrin and its formulated product: Contribution to mode of action research

Physiological responses of bacterial, fish, rat and human hepatoma cells to the technical cypermethrin (AS), cypermethrin-based plant protection product (PPP), and the major co-formulant (solvent) were compared. The endpoints included: bioluminescence, total protein content, activity of mitochondrial dehydrogenase and cytochrome P450 (CYP) enzymes CYP1A and CYP1B, and expression of several genes encoding different CYP enzyme isoforms. Toxicity of PPP was compared with the toxicity predicted using concentration addition model. Cypermethrin disturbs the activity of mitochondrial dehydrogenase. Induction of CYP1A1-, CYP1A2- and CYP1B1-associated activity was more pronounced in PPP than in cypermethrin treatment. The predominant biotransformation pathway of cypermethrin is related to Cyp3a1 induction. Deviations between observed and predicted toxicity of PPP indicate synergistic effects of cypermethrin and a solvent. In vitro cellular assays may serve as rapid pre-screening tool and provide for a good indication of mixture effects and prompt further in vivo testing of PPPs when really needed.     

Use of the dog as non-rodent test species in the safety testing schedule associated with the registration of crop and plant protection products (pesticides): present status

The results from a survey of the expert information that is publicly accessible on the use of the dog as test species during the regulatory evaluation of agricultural chemicals and pesticides are reported. Methods that are being used or considered in order to reduce the number of dogs used for this purpose are described. Regulatory evaluation aims at establishing threshold values for safe human exposure; it is based on no-observed-adverse-effect levels (NOELs) determined in animal studies. Current regulations require testing in two species, a rodent species (usually rat or mouse), and a non-rodent species (usually the dog). Subchronic (90-day) and chronic (12-month) repeated-dose feeding studies must be routinely conducted in dogs. This report first focuses on the results from a retrospective study analysing data on 216 pesticides kept on record by the Bundesinstitut für Risikobewertung, BfR (German Federal Institute for Risk Assessment), the competent regulatory authority in Germany. The study was sponsored and coordinated by SET, the German Foundation for the Promotion of Research on Replacement and Complementary Methods to Reduce Animal Testing (Stiftung zur Förderung der Erforschung von Ersatz-und Ergänzungsmethoden zur Einschränkung von Tierversuchen, Mainz) and conducted by the BfR. Since the data submitted for registration of a product is the property of the manufacturer, the study could only proceed with the collaboration of the German Association of Manufacturers of Agricultural Chemicals (Industrieverband Agrar, IVA). To ensure confidentiality, designated codes were used instead of the compounds proper names when the study was published. The results support two major conclusions. The use of the dog for the testing of pesticides is indeed necessary because the dog has proved to be the most sensitive species for about 15% of the compounds examined. However, chronic studies are only of limited value since they only provide essential information that cannot be obtained in sub-chronic studies in about 5% of cases. These conclusions are supported by several retrospective analyses using data on pharmaceutical drugs carried out in the context of the International Conference on Harmonisation of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH). Over 90% of drugs elicited no toxic symptoms in 12-month studies in dogs in addition to those that had been recorded previously in studies conducted for 90 or 180 days in dogs and rats. Another approach comparing the results from pre-clinical animal studies with clinical studies noted that animal studies predicted about 70% of the effects observed in volunteers, and in about 94% of cases the effects occurred in animal studies lasting not more than one month. Furthermore, the report summarises the current methods under consideration that could refine or reduce the use of dogs in toxicity testing: industrial data sharing and harmonisation of guidelines, in vitro methods, human studies, computational prediction models, and integrated testing approaches. The integrated Agricultural Chemicals Safety Assessment (ACSA) testing scheme, which is currently being developed in an international project initiated by the International Life Sciences Institute (ILSI, USA), is of particular relevance, since an ambitious attempt is being made to design a new comprehensive test framework incorporating modern scientific approaches and covering most aspects of current regulatory testing requirements. The ACSA project has access to the pesticide database of the US EPAs Office of Pesticide Programs (OPP). Preliminary results have confirmed the two major conclusions from the joint SET/BfR study conducted in Germany. Taking these results into account, it is recommended that the regulatory requirement for 12-month studies to be routinely carried out in dogs should be abandoned. While 90-day studies should be conducted in both rats and dogs, chronic studies should only take place in rats. If the dog is more sensitive than the rat in the 90-day study, an additional safety factor to the NOEL value obtained in the chronic rat study should be applied in order to set the threshold for safe human exposure, instead of conducting a 12-month study in dogs. This safety factor may be calculated from chronic NOEL data available in several pesticide databases. Chronic tests using dogs would then only be required if the test compound belongs to a new class of chemicals that has never been tested before. Thus, the report concludes that, according to current scientific knowledge, the routine 12-month studies in dogs are no longer required for agricultural chemicals and pesticides, and international regulations should be changed accordingly. Active international support of such measures is welcomed, from both an economical and an animal welfare perspective.Electronic Supplementary Material Supplementary material is available for this article at     

Predictive Acute Toxicity Tests With Pesticides.

Abstract By definition pesticides are biocidal products and this implies a probability that pesticides may be acutely toxic to species other than the designated target species. The ways in which pesticides are manufactured, formulated, packaged, distributed and used necessitates a potential for the exposure of non-target species although the technology exists to minimize adventitious exposure. The occurrence of deliberate exposure of non-target species due to the misuse of pesticides is known to happen. The array of predictive acute toxicity tests carried out on pesticides and involving the use of laboratory animals can be justified as providing data on which hazard assessment can be based. This paper addresses the justification and rationale of this statement.     

The ChemScreen project to design a pragmatic alternative approach to predict reproductive toxicity of chemicals

There is a great need for rapid testing strategies for reproductive toxicity testing, avoiding animal use. The EU Framework program 7 project ChemScreen aimed to fill this gap in a pragmatic manner preferably using validated existing tools and place them in an innovative alternative testing strategy. In our approach we combined knowledge on critical processes affected by reproductive toxicants with knowledge on the mechanistic basis of such effects. We used in silico methods for prescreening chemicals for relevant toxic effects aiming at reduced testing needs. For those chemicals that need testing we have set up an in vitro screening panel that includes mechanistic high throughput methods and lower throughput assays that measure more integrative endpoints. In silico pharmacokinetic modules were developed for rapid exposure predictions via diverse exposure routes. These modules to match in vitro and in vivo exposure levels greatly improved predictivity of the in vitro tests. As a further step, we have generated examples how to predict reproductive toxicity of chemicals using available data. We have executed formal validations of panel constituents and also used more innovative manners to validate the test panel using mechanistic approaches. We are actively engaged in promoting regulatory acceptance of the tools developed as an essential step towards practical application, including case studies for read-across purposes. With this approach, a significant saving in animal use and associated costs seems very feasible.     

The challenge of using read-across within the EU REACH regulatory framework; how much uncertainty is too much? Dipropylene glycol methyl ether acetate,an exemplary case study

The use of read-across of data within a group of structurally similar substances potentially allows one to characterise the hazards of a substance without resorting to additional animal studies. However the use of read-across is not without challenges, particularly when used to address the needs of a regulatory programme such as the EU REACH regulation. This paper presents a case study where a previously accepted read-across approach was used to address several data gaps in a REACH registration dossier but was subsequently rejected in part by the European Chemicals Agency (ECHA), resulting in the requirement to perform a developmental toxicity study in rodents. Using this case study, this paper illustrates some of the practical challenges faced when making use of read-across, particularly with respect to addressing the uncertainty associated with the use of read-across; showcasing the scientific justification and highlighting some of the potential implications/opportunities for future cases.     

Current in vitro methods in nanoparticle risk assessment: Limitations and challenges

Nanoparticles are an emerging class of functional materials defined by size-dependent properties. Application fields range from medical imaging, new drug delivery technologies to various industrial products. Due to the expanding use of nanoparticles, the risk of human exposure rapidly increases and reliable toxicity test systems are urgently needed. Currently, nanoparticle cytotoxicity testing is based on in vitro methods established for hazard characterization of chemicals. However, evidence is accumulating that nanoparticles differ largely from these materials and may interfere with commonly used test systems. Here, we present an overview of current in vitro toxicity test methods for nanoparticle risk assessment and focus on their limitations resulting from specific nanoparticle properties. Nanoparticle features such as high adsorption capacity, hydrophobicity, surface charge, optical and magnetic properties, or catalytic activity may interfere with assay components or detection systems, which has to be considered in nanoparticle toxicity studies by characterization of specific particle properties and a careful test system validation. Future studies require well-characterized materials, the use of available reference materials and an extensive characterization of the applicability of the test methods employed. The resulting challenge for nanoparticle toxicity testing is the development of new standardized in vitro methods that cannot be affected by nanoparticle properties.     

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands.Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent.Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed.Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.     

Proposal for a tiered approach to developmental toxicity testing for veterinary pharmaceutical products for food-producing animals.

In order to establish the safety of veterinary drug residues in human food, a number of toxicological evaluations are required, including the assessment of potential risks to development. In the light of the use of developmental toxicity testing for risk characterization, we evaluated whether conducting these tests in more than one species was redundant. Review of the published Summary Reports of recommendations of the EU Committee for Veterinary Medicinal Products supplemented with data from the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reports on veterinary drug residues in food identified 120 compounds, of which 105 had teratogenicity information in more than one species. The analysis of these compounds, representing a variety of chemical and pharmacological classes, provided justification for consideration of the use of a tiered approach for developmental toxicity evaluation of veterinary drugs for food-producing animals. The tiered approach begins with developmental toxicity testing in a rodent species, preferably the rat. If teratogenicity is observed, no testing in a second species would be required, except under specific circumstances where the ADI is determined based on the NOEL from this study. If a negative or an equivocal result for teratogenicity were observed in the rodent, then a developmental test in a second species, preferably the rabbit, would be conducted. The tiered approach provides thorough hazard identification, based on the use of a second species for compounds negative for teratogenicity in the rodent, and maintains maximum public protection based on the extremely low potential for human exposure to these compounds, while making a genuine attempt to limit unnecessary animal testing.     

Human exposure to synthetic endocrine disrupting chemicals (S-EDCs) is generally negligible as compared to natural compounds with higher or comparable endocrine activity. How to evaluate the risk of the S-EDCs?

Theoretically, both synthetic endocrine disrupting chemicals (S-EDCs) and natural (exogenous and endogenous) endocrine disrupting chemicals (N-EDCs) can interact with endocrine receptors and disturb hormonal balance. However, compared to endogenous hormones, S-EDCs are only weak partial agonists with receptor affinities several orders of magnitude lower. Thus, to elicit observable effects, S-EDCs require considerably higher concentrations to attain sufficient receptor occupancy or to displace natural hormones and other endogenous ligands.Significant exposures to exogenous N-EDCs may result from ingestion of foods such as soy-based diets, green tea and sweet mustard. While their potencies are lower as compared to natural endogenous hormones, they usually are considerably more potent than S-EDCs.Effects of exogenous N-EDCs on the endocrine system were observed at high dietary intakes. A causal relation between their mechanism of action and these effects is established and biologically plausible. In contrast, the assumption that the much lower human exposures to S-EDCs may induce observable endocrine effects is not plausible. Hence, it is not surprising that epidemiological studies searching for an association between S-EDC exposure and health effects have failed.Regarding testing for potential endocrine effects, a scientifically justified screen should use in vitro tests to compare potencies of S-EDCs with those of reference N-EDCs. When the potency of the S-EDC is similar or smaller than that of the N-EDC, further testing in laboratory animals and regulatory consequences are not warranted.     

Challenges to tradition—Environmental toxicology in the 1980s

Literature data on the toxicity of chemicals to fish, a daphnid, and a mammal were reviewed to form a basis for efficiently determining chronic no-effect levels for many chemicals as may be required by law. The principal measurements used in statistically determining chronic no-effect levels for chemicals are the acute LC₅₀, the maximum acceptable toxicant concentration (MATC), and the application factor (AF). Equations for these measurements were developed to help in predictive calculation of AFs, MATCs and LC₅₀'s for combinations of related and unrelated organisms (fish and Daphnia) and chemicals. None of these methods are entirely reliable but are useful for early assessment of chronic toxicity to determine whether extensive testing appears necessary. The degree of predictability increases with the use of shortened or short-life-cycle chronic toxicity data.     

Qualification of a non-animal vaginal irritation method admitted as nonclinical assessment model (NAM) in the Incubator Phase of the United States Food and Drug Administration (US FDA) Medical Devices Development Tool (MDDT)

The U.S. Food and Drug Administration (FDA) Center for Devices and Radiological Health (CDRH) classifies personal lubricants as Class II medical devices. Because of this status and the nature of body contact common to personal lubricants, CDRH reviewers routinely recommend a standard biocompatibility testing battery that includes: an in vivo rabbit vaginal irritation (RVI) test; an in vivo skin sensitization test, such as the guinea pig maximization test (GPMT); and an in vivo acute systemic toxicity test using mice or rabbits. These tests are conducted using live animals, despite the availability of in vitro and other non-animal test methods that may be suitable replacements. The only test included in the biocompatibility battery currently conducted using in vitro assay(s) is cytotoxicity. FDA's recently launched Predictive Toxicology Roadmap calls for the optimization of non-animal methods for the safety evaluation of drugs, consumer products and medical devices. In line with these goals, a Consortium comprising the Institute for In Vitro Sciences, Inc. (IIVS), industry, the Consumer Healthcare Products Association (CHPA), and the PETA International Science Consortium (PETA-ISC) is qualifying the use of an in vitro testing method as replacement for the RVI test. Participating companies include manufacturers of personal lubricants and those interested in the advancement of non-animal approaches working collaboratively with the FDA CDRH to develop an in vitro testing approach that could be used in place of the RVI in pre-market submissions. Personal lubricants and vaginal moisturizers with diverse chemical and physical properties (e.g., formulation, viscosity, pH, and osmolality) in their final undiluted form will be the focus of the program. In vitro vaginal irritation data generated using commercially available human reconstructed vaginal tissue model(s) will be paired with existing in vivo RVI data and analyzed to develop a Prediction Model for the safety assessment of these products. This research plan has been accepted into the FDA CDRH Medical Device Development Tools (MDDT) program as a potential non-clinical assessment model (NAM). The proposed NAM aligns with the goals of the recently launched FDA Roadmap to integrate predictive toxicology methods into safety and risk assessment with the potential to replace or reduce the use of animal testing.