Authors: Simona Panzacchi, Ramazzini Institute (panzacchis@ramazzini.it), Daria Sgargi, Ramazzini Institute (sgargid@ramazzini.it) and Daniele Mandrioli, Ramazzini Institute (daniele.mandrioli@protonmail.com)
Last update: 05/02/2026
The innovative principle involved in the SPRINT project was the three-tiered component-based approach used to evaluate the effects of individual pesticides using standard (European Food Safety Authority [EFSA] tests) and innovative tests (the New SPRINT Indicators).
- Tier 1 prioritized the top 10 pesticides for testing based on occurrence and risk data.
- Tier 2 used in vitro tests, to assess different standard and innovative mechanisms of toxicity, including cytotoxicity (cell damage) and genotoxicity (DNA damage).
- Tier 3 involved in vivo studies in rats and mice differentiated by sex.
In general, the pesticides tested showed adverse effects on the endocrine system, liver, kidneys, microbiome, and behavioral alterations, even at doses corresponding to levels currently considered safe for humans (or without adverse effects in animals). Pesticide mixtures proved to be consistently more detrimental, consistently showing additive (non-synergistic) and linear dose-responses. The new models used (the New SPRINT Indicators) proved to be sensitive and relevant for different toxicological endpoints of interest.
In the following sections, the users will find a description of the tool and the information it provides for three different end-user groups. We highly recommend that users study this information before going to the detailed results. From the left bottom menu, users can access the details and summaries of methods and results for each Tier.
Description for General Public
This tool was constructed to collect all the information and evidence that emerged in the several experiments conducted within the SPRINT project to gain insight into the toxicity of selected pesticides.
The user can follow the phases of the research performed:
The first phase (Tier 1) was based on available data collected in the SPRINT project on over 200 pesticides, assessing and prioritizing the 20 most concerning pesticides.
The second phase (Tier 2) used standard in vitro experiments together with new models developed by the SPRINT project (the New SPRINT Indicators) to test the 10 most concerning pesticides and mixtures.
In the third phase (Tier 3), experimental animal studies were used together with new studies created for the SPRINT project (the New SPRINT Indicators) to test the top 3 pesticides of concern and their mixtures.
Description for Policy Makers
In this Tool we present the results of the assessment of the effects of pesticides and their mixtures on human health performed in the SPRINT project. The research was designed following the component-based tiered approach based on the EFSA guidelines for combined exposure to multiple chemicals (EFSA, 2019), to assess the effects of individual pesticides and mixtures, and to investigate new scientific indicators (the New SPRINT Indicators) that could be useful in the future for risk assessment evaluation.
The user can follow the phases (Tiers) of the research performed:
In Tier 1 based on the available data from the SPRINT survey 2021 on over 200 pesticides and in silico modelling, the top 20 pesticides of concern were assessed and prioritized for in vitro testing based on occurrence and weighted hazard quotient (wHQ).
In Tier 2, standard in vitro models (EFSA tests) along with new models (the New SPRINT Indicators) were used to test the Top 10 pesticides and mixtures of concern.
In Tier 3, standard in vivo models (EFSA tests) along with new models (the New SPRINT Indicators) were used to test the Top 3 pesticides of concern and their mixtures.
The results showed endocrine disruptive effects, liver toxicity, kidney toxicity, microbiome and behavioural alterations, even at doses corresponding to levels that are currently considered safe in humans (or without adverse effects in animals, i.e. between ADI - Acceptable Daily Intake and NOAEL - No Observed Adverse Effect Level).
All the pesticides that have been tested in Tier 2 and 3 showed at least one positive signal. Pesticide mixtures proved to be consistently more detrimental, consistently showing additive (non-synergistic) and linear dose-responses, confirming that a component-based approach is predictive and adequate for hazard and risk assessment of mixtures.
The new models used (the New SPRINT Indicators), both in vitro and in vivo, proved to be sensitive and relevant for different toxicological endpoints of interest.
Limitations / Disclaimer
The mechanistic information provided by the New SPRINT Indicators could inform hazard assessment on standard (in vitro kidney toxicity, in vivo behavioural effects) and new (in vivo microbiome effects) endpoints, but further validation is required for their use for risk assessment and pre-market evaluations.
Description for Scientists
Background / How it works
Here we present the results of the assessment of the effects of pesticide mixtures on human health investigated in the SPRINT project. A Tiered approach, from simple assessments at low-tier to more complex assessments at high-tier, has been implemented based on the EFSA guidelines for combined exposure to multiple chemicals using a component-based approach (EFSA, 2019).
What it offers
The component-based tiered approach was used to assess the effects of individual pesticides and mixtures:
- In Tier 1, based on the available data from the SPRINT Case Study Sites (CSS) on over 200 pesticides and in silico modelling, the top 20 pesticides of concern were assessed and prioritized for in vitro testing based on occurrence and weighted Hazard Quotient (wHQ). A systematic procedure was developed to prioritise pesticides for toxicology testing using a systematic stepwise procedure based on occurrence (exposure), hazard, and risk information. The decision to select the Top 10 pesticides to be tested for the in vitro studies was through occurrence via the SPRINT CSS data, and by toxicity, based on updates of the EFSA guidance on Acceptable Daily Intake (ADI) and Acceptable Operator Exposure Level (AOEL).
- In Tier 2, standard in vitro models such as cytotoxicity assays and micronuclei assays (EFSA tests) along with new models like kidney cells assays and organoid assays (the New SPRINT Indicators) were used to test the Top 10 pesticides and mixtures of concern. This battery of assays allowed to show cytotoxic and genotoxic effects in a linear and additive dose-response (no synergistic effects observed), along with alterations of piglet organoids and human kidney cell line viability and transcriptome.
- In Tier 3, standard in vivo models such as a 13-weeks toxicity study in rats exposed from prenatal life (EFSA tests) along with new models like a series of behavioural and microbiome studies in mice (the New SPRINT Indicators) were used to test the Top 3 pesticides of concern (glyphosate, acetamiprid, tebuconazole) and their mixtures. Two other analyses based on samples derived from the main studies were performed: microbiome analyses in rats and kidney microscopy and morphometric analyses in rats and mice. The assays addressed endocrine disruptive effects, liver toxicity, kidney toxicity, microbiome and behavioural alterations.
All the pesticides that have been tested in Tier 2 and 3 showed at least one positive signal. The results showed endocrine disruptive effects, liver toxicity, kidney toxicity, microbiome and behavioural alterations, even at doses corresponding to levels that are currently considered safe in humans (or without adverse effects in animals, i.e. between ADI - Acceptable Daily Intake and NOAEL - No Observed Adverse Effect Level).
Pesticide mixtures proved to be consistently more detrimental, consistently showing additive (non-synergistic) and linear dose-responses. We did not find any clear sign of non-additivity (synergism or antagonism) in the mixtures tested. Therefore, in vitro and in vivo human toxicology confirm that a component-based approach is predictive and adequate for hazard and risk assessment of mixtures.
The new models used (new SPRINT indicators), both in vitro and in vivo, proved to be sensitive and relevant for different toxicological endpoints of interest. The information provided by the new SPRINT indicators has proven useful for assessing the risks associated with new effects.
Limitations / Disclaimer
The mechanistic information provided by the New SPRINT Indicators could inform hazard assessment on standard (in vitro kidney toxicity, in vivo behavioural effects) and new (in vivo microbiome effects) endpoints, but further validation is required for their use for risk assessment and pre-market evaluations.
Moreover, technical limitations (such as solubility of the pesticide mixture components and the number of components that can be tested in a single run) represent important challenges for adequate modelling and dose-response in vitro and in vivo human toxicological studies of mixtures.