Registration Dossier

Toxicological information

Specific investigations: other studies

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
toxicogenomics
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The aim of this study was to examine the effect of 2-PO on hepatic gene expression using a toxicogenomics (TGX) approach in an attempt to identify modes of action (MOA) underlying possible hepatotoxicity. In a more biased approach, genes related to carcinogenesis were also examined. Male and female Sprague-Dawley rats were exposed to 2-PO by inhalation at concentrations of 50 ppm, 150 ppm and 300 ppm. The rats were exposed to 2-PO for 6 hours per day, 5 days per week, over a 90-day period. To assess recovery or delayed occurrence of toxicity, additional groups (control and high concentration) were exposed for the same period and kept for an observation period of 4 weeks following exposure (recovery groups). These samples were examined separately (male animals only n = 8 per group; control vs 300 ppm 2-PO) once the results of the main study were known.
Whole Genome Expression was examined using Agilent Microarrays. Genespring™ software was used to identify differentially expressed genes in the 2-PO-dosed animals relative to control samples (p < 0.05; fold change > 1.5). An unbiased analysis using Ingenuity Pathway Analysis (IPA™) software was used to identify genes over-represented in toxicologically-relevant pathways. In addition, a biased approach to examine changes in specific toxicity pathways was also conducted using the IPA™ Path Explorer function. Expression of genes related to carcinogenicity was also examined using the IPA™ software.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
other: toxicogenomics
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data
Route of administration:
inhalation
Vehicle:
not specified
Details on exposure:
not specified
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6 h / day over a 90-day period
Frequency of treatment:
5 days / week
Post exposure period:
4 weeks (males only; controls and high dose group)
Dose / conc.:
50 ppm
Remarks:
target concentration
Dose / conc.:
150 ppm
Remarks:
target concentration
Dose / conc.:
300 ppm
Remarks:
target concentration
No. of animals per sex per dose:
- main study: 10 / sex / dose
- recovery groups: 8 male animals (control and high dose group)
Control animals:
yes
Details on study design:
Male and female Sprague-Dawley rats were exposed to 2-PO by inhalation at concentrations of 50 ppm, 150 ppm and 300 ppm. The rats were exposed to 2-PO for 6 hours per day, 5 days per week, over a 90-day period. To assess recovery or delayed occurrence of toxicity, additional groups (control (Ctrl) and high concentration) were exposed for the same period and kept for an observation period of 4 weeks following exposure (recovery groups). These samples were examined separately (male animals only n = 8 per group; control vs 300 ppm 2-PO) once the results of the main study were known. Liver samples were snap frozen in liquid nitrogen, and stored frozen (circa -70°C) prior to analysis. Samples were shipped to CXR Biosciences on dry ice, received intact on 8th June 2016 and stored at CXR Biosciences at (circa -70°C) prior to RNA extraction and analysis. Whole Genome Expression was examined using Agilent Microarrays. Genespring™ software was used to identify differentially expressed genes in the 2-PO-dosed animals relative to control samples (p < 0.05; fold change > 1.5). An unbiased analysis using Ingenuity Pathway Analysis (IPA™) software was used to identify genes over-represented in toxicologically - relevant pathways. In addition, a biased approach to examine changes in specific toxicity pathways was also conducted using the IPA™ Path Explorer function. Expression of genes related to carcinogenicity was also examined using the IPA™ software.
Examinations:
The aim of this study was to examine the effect of 2-PO on hepatic gene expression using a TGX approach in an attempt to identify modes of action (MOA) underlying possible hepatotoxicity and genes / pathways related to carcinogenesis.
To this end, both biased and unbiased TGX analysis of gene expression data was performed. The unbiased analysis focused on identifying the most statistically significant functions over-represented in the rat liver gene signature lists. The biased approach (biased analysis) using the IPA™ Path Explorer function, examined changes in specific toxicity and carcinogenicity-related pathways.
- Sample Source and General Experimental Approach:
Liver samples were snap frozen in liquid nitrogen, and stored frozen (circa -70°C) prior to analysis. Samples were shipped to CXR Biosciences on dry ice, received intact on 8th June 2016 and stored at CXR Biosciences at (circa -70°C) prior to RNA extraction and analysis.

RNA was extracted from the frozen rat liver samples, purified, checked for quality (QC) (Agilent Tapestation QC). Samples then underwent one colour (Cy3) labelling and hybridization of RNA on Agilent 8 x 60 K Rat Whole Genome Expression Microarrays (56 samples in total). The microarray analyses were performed over a single contiguous run.

- RNA isolation:
Tissues were disrupted using a Polytron™ (Kinematic, Switzerland # 9115099) and extracted into Tri reagent (Sigma, T9424-100 mL) using a modified method reported by Chomczynski and Sacchi (1987). Total RNA was purified using RNeasy mini columns (Qiagen GmbH # 75144) according to Standard Operating Procedure (SOP) CXR –LMS-TRAP-026.

- Quality control of RNA using the Agilent Bioanalyser:
RNA integrity (40 samples) was checked using the Agilent Tape station and the RNA screentape according to CXR-LMS-TRAP-029. The screentape was used for RNA with a concentration of between 25 ng/μL and 500 ng/μL. It was necessary (due to RNA yield) to dilute the RNA in RNase-free water for the screentape.

- Microarrays:
Agilent Whole Rat Genome Oligo Microarray slides 8 x 60k (G4853A) were hybridised, washed and then scanned on an Agilent Microarray Scanner according to the CXR – LMS- TRAP-015.

- Microarray experimental structure:
The microarray analysis involved Cy3 labelled (one colour) RNA from livers collected from male (M) and female (F) rats exposed (inhalation) to 50 ppm, 150 ppm or 300 ppm 2-Pentanone oxime. The layout for the samples was designed to minimize inter- and intra-array variability and eliminate confounding. This layout accounted for all liver samples that were received at CXR Biosciences from TNO.

- Probe Labelling, Microarray Hybridisation and Scanning:
Total RNA (50 ng) was labelled with Cyanine 3-CTP (Cy3), a fluorescent dye that enables visualisation and quantification when scanned, prior to microarray hybridisation using the Agilent Low Input Quick Amp Labelling Kit One Colour (Agilent# 5190-2305), according to SOP CXR –LMS-TRAP-007. The isolated, labelled RNA sample from each animal was applied onto an Agilent 8 x 60K Whole Rat Genome Oligo Microarray slide (G4853A). Hybridisation to the arrays was performed in a designated hybridisation oven (Robbins scientific) at 65°C for 17 h. The microarrays were hybridised, washed and then scanned using an Agilent Microarray Scanner according to the SOP CXR TRAP-015.

- Microarray Data Processing and Bioinformatics:
Images from the scanner were processed using Agilent Feature Extraction Software v10.7.3.1. Agilent Genespring™ GX 13.1 software was used to define a list of significantly altered genes (the DEG list). DEG lists of significantly altered genes (p < 0.05), filtered to remove low intensity genes (filtered on expression level, -0.3 – 0.3 (intensity units / lower and upper cut-off)), were generated. A 2-way Analysis of variance (ANOVA) was applied in order to ascertain the influence of the two independent categorical variables “dose (exposure)” and sex” as one continuous dependent variable. Following ANOVA, fold change (> 1.5 fold) calculation and p-value computation was applied (asymptotic). Multiple correction testing was performed using Benjamini-Hochberg false discovery rate (FDR). In this study the combined application of a moderate fold change threshold (FC > 1.5) and a stringent FDR threshold generated a reliable set of biological processes identified as differentially regulated. Application of p < 0.05 is an essential pre-requisite to reduce the number of false positives. For ease of reference, the gene lists were named with the test compound and dose (exposure) vs control for each iteration. These lists were compared to ascertain correlation.
Evaluation of differentially expressed genes by treatment at each time point was performed using Genespring. If appropriate, unfocused Pathway over-representation analysis of DEGs (if any) was then carried out utilising Ingenuity Pathways Analysis™ (IPA™). An unbiased (unfocused) global approach was used to analyse the differentially expressed genes and pathways as determined by IPA™; the pathways with the greatest statistical significance were listed in rank order. These analyses were then used to examine any dose response relationship. In order to address the issues of hepatotoxicity and / or carcinogenicity and if appropriate, focused analysis using IPA™, was carried out to examine the following specific pathways:
Liver proliferation
Liver necrosis / apoptotic cell death
Cell Cycle
Inflammation / lipid metabolism
Haem / Iron metabolism

These pathways within IPA™ were selected on the basis of trends seen in the IPA™ pathways unbiased analyses, and based on alternative Modes of Action for liver effects and/or potential pathways of interest for this class of compound. Liver tumours generally occur at dose levels that also produce cytotoxicity. Alternative modes of carcinogenic action may involve cytotoxicity followed by regenerative cell proliferation or growth promotion of cells in the absence of significant cytotoxicity (i.e. mitogenesis).

IPA™ was also used to examine upstream regulators (transcription factors / regulators etc.) to shed further light on biological plausibility by examining which transcriptional regulators and their targets may control expression, and how these upstream molecules may regulate one another. The goal of the IPA Upstream Regulator analysis is to identify the cascade of upstream transcriptional regulators that can explain the observed gene expression changes in the dataset. The Z-score indicates the extent to which downstream molecules are over-represented in the respective gene signatures and the polarity of these changes determines whether an activation state or inhibitory status is applied. A Z-score greater than 2 is considered of significant magnitude to be worthy of note and indicates either activation of inhibition in terms of the observed response of the downstream genes.

In the IPA™ analyses the p values represent the likelihood that the association between the canonical pathways and the genes in the signature lists is due to random chance. The p value is calculated with a right tailed Fishers Exact test. The ratio represents the number of genes in a canonical pathway that are found in the signature list divided by the total number of genes in the pathway. A Z-score algorithm is used to determine the overall activity score for each of the given pathways. The canonical pattern is defined using an Ingenuity derived algorithm which takes into account the activation state of one or more key genes, when a given pathway is shown to be activated. These genes’ causal relationship with one another and interacting molecules (based on the Ingenuity literature database) generates an overall pattern of activity of genes, molecules and end points in the given signalling cascade (canonical pathway). This then represents the canonical or expected pattern for each eligible canonical pathway and is used for scoring the inputted dataset resulting in a prediction of overall activation or inhibition of the pathway (reference: IPA™). In the relevant figures bars are coloured according to the predicted overall activation: ORANGE = activated / BLUE = inhibited / GREY or WHITE = no activity pattern available for analysis. The Ratio is presented on the right-hand axis for each indicated pathway, and values are reflected by the orange dots relative to the number of genes in that pathway. For example, the Phosphatidylglycerol signaling pathway had a Ratio = 0.15, meaning 15% of the total genes in that pathway were differentially expressed through exposure to 150 ppm 2-PO. The colour of the bars for each pathway represents the Z-score, and those above the “threshold” line reflect statistically significant pathways.
Positive control:
no
Details on results:
- Exposure of male rats to 2-PO produced increased expression of genes involved in xenobiotic metabolism (at 150 ppm 2-PO and 300 ppm 2-PO), namely GSTa3, GSTm (approximately 21 fold up-regulated at 150 ppm,) and UGT (2 - 3 fold up-regulated at 150 ppm).
- Analysis of canonical and toxicological pathways by IPA™ demonstrated possible involvement of CAR and PXR in the hepatic response to 2-PO, however the absence of an increase in CYP2B expression makes this equivocal. Moreover, histopathology carried out at the end of 90-day exposure (not part of this report) did not show any indication of hypertrophy and hyperplasia, the hallmark of CAR and CXR activation.
- Analysis using IPA™ revealed involvement of peroxisome proliferator-activated receptor alpha (PPAR-α) and gamma (PPAR-gamma) in response to 2-PO (ADIPOR2, ACSL5, HADAHB, PNPLA2 were all down-regulated). A lack of CYP4A induction suggests a lack of conventional activation of PPAR-α.
- The observations made for CAR, CXR and PPAR pathways are not prototypical response from non-genotoxic carcinogens (NGCs). This finding is corroborated with the biological findings with lack of cell proliferation and hypertrophy. Therefore, there is no strong evidence of NGC mode of action for 2-PO through these pathways.
- In the absence of CYP1A1 induction there is no strong evidence for Aryl hydrocarbon receptor (AhR) activation.
- Changes in genes involved in cell cycle and proliferation were examined. The biggest change in a single gene involved in cell proliferation was increased expression of CDKN1A (p21/Cip1, 9 fold up-regulation) following exposure to 150 ppm and 300 ppm 2-PO. However, overall the fold-change and polarity of the gene expression changes were not consistent with a mitogenic response.
- These changes were not seen when the recovery group (2-PO 300 ppm vs. control) was examined. The gene expression shift back to baseline following recovery would suggest that the acute effects of 2-PO exposure are transient and dependent of duration of exposure and magnitude of dose.

Conclusion
In male rats exposure to 2-PO at 150 ppm or 300 ppm, resulted in dose-dependent increases in the expression of xenobiotic metabolizing enzymes (e.g. GSTs, UGTs). Genes involved in an oxidative stress response were similarly altered.

In contrast, there were no statistically significant differentially expressed genes observed at 50 ppm 2-PO versus control in male rat liver. In female rat liver, no genes were found to be differentially expressed at statistically significant levels (P<0.05) in response to exposure to 2-PO at all concentrations.

In male rat liver gene expression changes consistent with an inflammatory response and dyslipidemia occurred at 150 ppm and 300 ppm 2-PO. However, these changes are not associated with any pathological changes in liver observed after 90-days of exposure.

Biased analysis of the gene expression data from male rat liver showed that changes consistent with inhibition of cell proliferation occurred at the higher exposure levels of 2-PO (150 ppm & 300 ppm). This result is consistent with the histopathological findings for this study as there was no hyperplasia or hypertrophy of hepatocellular cells.

In summary, the changes induced in rat liver by 2-PO were induction of drug metabolising enzymes, oxidative stress, and inflammation as expected from exposure to high concentration of industrial chemicals. Although gene expression changes (a combination of p21 / CDKN1A and O6-MGMT up-regulation) indicated possible DNA damage, such findings are not corroborated with the genotoxicity information available for this molecule. Overall, this molecule is non-genotoxic. A 14-day inhalation study followed by comet assay demonstrated that 2-PO is not a genotoxic compound in animals. Further, no evidence was found for a mitogenic (cell proliferative) response. There is no strong evidence of CAR, PXR and AhR induction in male or female rats. This finding also corroborated with the histological findings in liver.
Examination of the recovery groups revealed that changes observed at high concentrations groups i.e. induction of drug metabolising enzymes, oxidative stress, and inflammation responses are reversible. The gene expression shift back to baseline following recovery would suggest that the acute effects of 2-PO exposure are transient and dependent of duration of exposure and magnitude of dose.

Description of key information

Toxicogenomic study (male & female rats):

- changes induced in rat liver were: induction of drug metabolising enzymes, oxidative stress, and inflammation; changes are transient (reversible within recovery group) and dependent of duration of exposure and magnitude of dose

Additional information

The aim was to examine the effect of 2-Pentanone oxime (2 -PO) on hepatic gene expression using a toxicogenomics approach in an attempt to identify modes of action underlying possible hepatotoxicity. In a more biased approach, genes related to carcinogenesis were also examined.

Male and female Sprague-Dawley rats were exposed to 2-PO by inhalation at concentrations of 50 ppm, 150 ppm and 300 ppm. The rats were exposed to 2-PO for 6 h per day, 5 days per week, over a 90-day period. To assess recovery or delayed occurrence of toxicity, additional groups (control and high concentration) were exposed for the same period and kept for an observation period of 4 weeks following exposure (recovery groups). These samples were examined separately (male animals only n = 8 per group; control vs 300 ppm 2-PO) once the results of the main study were known.

Whole Genome Expression was examined using Agilent Microarrays. Genespring™ software was used to identify differentially expressed genes in the 2-PO-dosed animals relative to control samples (p < 0.05; fold change > 1.5). An unbiased analysis using Ingenuity Pathway Analysis (IPA™) software was used to identify genes over-represented in toxicologically - relevant pathways. In addition, a biased approach to examine changes in specific toxicity pathways was also conducted using the IPA™ Path Explorer function. Expression of genes related to carcinogenicity was also examined using the IPA™ software.  In male rats exposure to 2-PO at 150 ppm or 300 ppm, resulted in dose-dependent increases in the expression of xenobiotic metabolizing enzymes (e.g. GSTs, UGTs). Genes involved in an oxidative stress response were similarly altered. In contrast, there were no statistically significant differentially expressed genes observed at 50 ppm 2-PO versus control in male rat liver. In female rat liver, no genes were found to be differentially expressed at statistically significant levels (P<0.05) in response to exposure to 2-PO at all concentrations. In male rat liver gene expression changes consistent with an inflammatory response and dyslipidemia occurred at 150 ppm and 300 ppm 2-PO. However, these changes are not associated with any pathological changes in liver observed after 90-days of exposure. Biased analysis of the gene expression data from male rat liver showed that changes consistent with inhibition of cell proliferation occurred at the higher exposure levels of 2-PO (150 ppm & 300 ppm). This result is consistent with the histopathological findings as there was no hyperplasia or hypertrophy of hepatocellular cells. 

In summary, the changes induced in rat liver by 2-PO were induction of drug metabolising enzymes, oxidative stress, and inflammation as expected from exposure to high concentration of industrial chemicals. Although gene expression changes (a combination of p21 / CDKN1A and O6-MGMT up-regulation) indicated possible DNA damage, such findings are not corroborated with the genotoxicity information available for this molecule. Overall, this molecule is non-genotoxic. A 14-day inhalation study followed by comet assay demonstrated that 2-PO is not a genotoxic compound in animals. Further, no evidence was found for a mitogenic (cell proliferative) response. There is no strong evidence of CAR, PXR and AhR induction in male or female rats. This finding also corroborated with the histological findings in liver from the 90-day inhalation study. Examination of the recovery groups revealed that changes observed at high concentrations groups i.e. induction of drug metabolising enzymes, oxidative stress, and inflammation responses are reversible. The gene expression shift back to baseline following recovery would suggest that the acute effects of 2-PO exposure are transient and dependent of duration of exposure and magnitude of dose.