Complementary Use of Read-Across and Other New Approach Methodology Approaches to Address Complex Scientific and Regulatory Challenges

Project Summary

When industry-standard read-across methods fall short, you need evidence that stands up to regulatory scrutiny. 

When a European authority proposed unjustified harmonized hazard classifications for nitroparaffins based on inappropriate read-across, a specialty chemicals manufacturer needed a comprehensive scientific assessment that would meet the highest regulatory standards. The challenge: demonstrate why structural similarity alone doesn’t justify read-across when substantial toxicological differences exist.  

Integral assembled a multidisciplinary team to evaluate multiple lines of evidence—including cutting-edge New Approach Methodologies (NAMs)—proving that regulatory classification decisions must be grounded in robust scientific data, not simplified assumptions. 

Challenge

Chemical structure similarity doesn’t necessarily equal toxicological similarity—but regulators often assume it does. 

Our client faced a significant regulatory challenge: European authorities proposed harmonized classification and labeling for nitroethane (NE) and 1-nitropropane (1NP) based solely on read-across from nitromethane (NM). Despite these three primary nitroparaffins sharing a common functional group, the proposal ignored: 

• Existing negative cancer bioassays for NE and 1NP 

• A negative developmental and reproductive toxicity (DART) study for 1NP 

• Substantial differences in physicochemical properties, metabolism, and biological effects. 

The stakes were high. An inappropriate classification could restrict product use, increase costs, and set a concerning precedent for oversimplified read-across approaches—all while disregarding chemical-specific data that demonstrated safety. 

Without a comprehensive, scientifically defensible response addressing modern regulatory frameworks like the European Chemicals Agency (ECHA) Read-Across Assessment Framework (RAAF), our client risked: 

• Unjustified restrictions on safe chemical products 

• Unnecessary animal testing to “fill” data gaps that didn’t exist. 

Our Role

Integral served as the scientific authority demonstrating why evidence-based assessment must replace assumptions. 

Our multidisciplinary toxicology team designed and executed a comprehensive weight-of-evidence evaluation in accordance with RAAF principles. Rather than simply arguing against the proposed classification, we built a proactive scientific case demonstrating exactly where and why the regulator’s read-across approach failed. 

We coordinated with ScitoVation to incorporate novel transcriptomics and high-throughput toxicokinetics (HTTK) data—demonstrating how NAMs can provide critical mechanistic insights that complement and support read across. 

What We Delivered

We delivered a systematic, multi-evidence stream assessment proving that read-across should be based on more than just structural similarity. Together with ScitoVation, we used NAMs to provide mechanistic insights and fill data gaps without animal testing. 

Integral’s analysis evaluated five complementary lines of evidence: 

Structural Similarity
We confirmed that NM, NE, and 1NP share a common nitro group but differ in alkyl chain length. While structural similarity is required for read-across, it is not alone a sufficient justification. 

Physicochemical Properties
Our analysis revealed that NE and 1NP are much more lipophilic than NM. HTTK modeling demonstrated that, due to lipophilicity differences, the nitroparaffins are expected to have different systemic distribution and local tissue metabolism rates—undermining a fundamental assumption of the read-across hypothesis. 

Toxicokinetic Properties
We determined that while all three compounds may form nitrite, they are expected to produce distinctly different aldehyde metabolites (formaldehyde, acetaldehyde, and propionaldehyde, respectively). Critically: 

• The different aldehyde metabolites have distinct toxicological profiles 

• Formation of these different aldehyde metabolites has not been consistently demonstrated in vivo 

• The compounds differ quantitatively in their propensity to form shared metabolites (nitrite). 

Toxicodynamic Properties
Integration of transcriptomics data from an in vitro study in rat liver cells revealed minimal overlap in dysregulated gene expression and gene ontology profiles among the three chemicals. The dysregulated genes and pathways showed no patterns characteristic of carcinogens or reproductive toxicants—directly challenging the mechanistic basis for read-across. 

Toxicological Properties
Our comprehensive review of existing studies demonstrated: 

• Carcinogenicity: NM’s positive findings in rodents were questionable (tumors within historical control ranges), while NE and 1NP showed negative results in sufficiently robust studies 

• DART: Effects of NM occurred only at doses exceeding the maximum tolerated dose alongside severe maternal toxicity, while 1NP showed no developmental effects in a guideline-compliant study. 

Throughout the analysis, we systematically evaluated each line of evidence using the RAAF assessment elements, documenting exactly where the proposed read-across failed to comply with regulatory guidance. 

The Result

Our client gained a scientifically bulletproof defense demonstrating that existing animal data, supported by NAMs evidence, enables appropriate, chemical-specific classifications. 

Our comprehensive assessment proved that the three nitroparaffins should not be grouped for read-across. The evidence showed: 

• No common mechanism of action linking the compounds for carcinogenicity or DART endpoints 

• Transformation to different compounds with distinct toxicological profiles 

• Substantial differences across physicochemical, toxicokinetic, toxicodynamic, and toxicological properties. 

This work provided our client with: 

Immediate Regulatory Defense: Comments submitted to the ECHA Committee for Risk Assessment demonstrating that the proposed classifications were scientifically unjustified and inconsistent with ECHA’s own RAAF principles. 

Precedent for NAMs Integration: Clear documentation of how NAMS, including in vitro transcriptomics assays and HTTK modeling, can fill critical data gaps without additional animal testing—advancing both scientific rigor and ethical alternatives. 

Protection of Existing Data: Validation that high quality chemical-specific negative studies for NE and 1NP should not be replaced with inappropriate read-across to positive NM data, preventing unnecessary restrictions. 

Broader Industry Impact: A case study demonstrating that regulatory decisions must be based on comprehensive evidence evaluation, not on oversimplified structural comparisons, which is being prepared for submission to a peer-reviewed journal. 

The analysis reinforced a critical principle: when adequate chemical-specific data exist, read-across is not just inappropriate—it undermines scientific assessment by replacing evidence with assumptions.