2-propylheptan-1-ol

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline available

Principles of method if other than guideline:

Analysis of the metabolite profile (metabolome) in plasma of male and female rats treated with the test substance for 28 days.
Plasma was taken from fasted rats after 28-day treatment. The plasma metabolome, i.e. 228 endogenous plasma components such as carbohydrates, amino acids, hormones, fatty acids, etc., was investigated in regard to changes relative to the control group. 44 of the components were determined in by two independent methods so that the overall number of measurements is 272.
The specific interest of this study was the evaluation of the biological similarity of the two test substances 2-Propylheptanol and Bis-(2-propylheptyl)phthalate regarding their systemic toxicity.

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

other: metabolome analysis (228 endogenous plasma components)

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

other: gavage (2-Propylheptanol)/feed (DPHP)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

200 mg/kg bw/day

Remarks:

CAS 10042-59-8

Dose / conc.:

500 mg/kg bw/day

Remarks:

CAS 10042-59-8

Dose / conc.:

3 000 ppm

Remarks:

CAS 53306-54-0

Remarks:

CAS 53306-54-0

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

BLOOD SAMPLING:
- Blood samples for metabolome analysis were taken by puncturing the retrobulbar venous plexus on study day 29 from overnight fasted animals under isoflurane anesthesia. From each animal, 1 mL of blood was collected. The samples were centrifuged and the plasma was separated. All samples were stored covered with a N2 atmosphere and then stored at –80°C before analysis.

METABOLITE PROFILING:
The plasma metabolome was examined using GC-MS and LC-MS/MS techniques. Briefly, two types of mass spectrometry analysis were applied to all samples: GC-MS (gas chromatography-mass spectrometry) and LC-MS/MS (liquid chromatography-MS/MS) were used for broad profiling, as described in van Ravenzwaay et al. (2007). Proteins were removed from plasma samples by precipitation. Subsequently polar and non-polar fractions were separated for both GC-MS and LC-MS/MS analysis by adding water and a mixture of ethanol and dichloromethane. For GC-MS analysis, the non-polar fraction was treated with methanol under acidic conditions to yield the fatty acid methyl esters derived from both free fatty acids and hydrolyzed complex lipids. The non-polar and polar fractions were further derivatized with O-methyl-hydroxylamine hydrochloride and pyridine to convert oxo-groups to O- methyl-oximes and subsequently with a silylating agent before analysis (Roessner et al., 2000). For LC-MS/MS analysis, both fractions were reconstituted in appropriate solvent mixtures. HPLC was performed by gradient elution using methanol/water/formic acid on reversed phase separation columns. Mass spectrometric detection technology was applied which allows target and high sensitivity MRM (Multiple Reaction Monitoring) profiling in parallel to a full screen analysis (patent application 2003073464). Absolute quantification was performed by means of stable isotope-labelled standards. For all metabolites, changes were calculated as the ratio of the mean of metabolite levels in individual rats in a treatment group relative to mean of metabolite levels in rats in a matched control group (time point, dose level, sex).

METAMAP®TOX EVALUATION
The sex- and day-stratified heteroscedastic t-test ("Welch test") was applied to compare metabolite levels of dose groups with respective controls. A significance of p < 0.05 was applied.
On the basis of a significance level of 0.05 (“false positive” rate) 5% significant metabolite changes can be expected by chance. Therefore, up to 5% significantly changed metabolites, the metabolome is considered as not affected by the test compound.
Test substance related changes in the metabolome were analyzed as follows:
1) Analysis of specific metabolic changes for each dose group. The percentage of significantly changed metabolites on the basis of all 272 metabolites minus metabolites with the status NA. Here also the profile strength is addressed, which is a parameter calculated in the treatment correlation. This parameter represents the rounded down average of absolute medians of t-values and does not only include the absolute number of significantly changed metabolites, but also the magnitude of the respective changes. With the analytical methods used (polar and non-polar fraction in GC-MS and LC-MS/MS) a metabolite can be detected in more than one phase leading to metabolite doublettes.
2) Using an established algorithm, the similarity of the test compound metabolic profile with the predefined patterns covering different modes of action in MetaMap®Tox was determined and evaluated by an expert panel, so-called “pattern ranking”. The ranking is based on the medians of the cosinus correlations for each pattern (median r). Each pattern has been created with a certain p-value (between 0.05 and 0.2).
The outcome of this assessment is one of four defined categories: The metabolite changes match a certain mode of action pattern (“match”, >90% of the metabolites are conform with the pattern), the metabolomic change is weakly associated with a mode of action (“weak match”, >75-90% of the metabolites are conform with the pattern), no conclusion is possible (“equivocal”, 50-75% of the metabolites are conform with the pattern) or the metabolic change does not match with a mode of action pattern (“mis-match”, less than 50% of the metabolites are conform with the pattern). Patterns containing metabolites with the status NA as anchor metabolites cannot be evaluated (for pattern conformity anchor metabolites of the pattern - if defined for the pattern- must be significantly changed by a test substance in the correct direction). Further reference can be found in Looser et al., 2005; Strauss et al., 2009, Kamp et al., 2012, and van Ravenzwaay et al., 2007, 2010 and 2014.
3) Comparison with the entire metabolome of reference compounds, called “treatment correlation” using Pearson and Spearman correlations. In order to assess the size and relevance of a correlation coefficient a reference distribution of correlation coefficients was derived by calculating all pairwise coefficients of the whole data base stratified by sex (male/female) and dose (high/low). As each stratum comprises approximately 500 treatments (t-profiles) the quantiles of each reference distribution are based on approximately 130000 r-values/stratum.
Based on these analyses, a threshold value of 0.40 for male animals and 0.50 for female animals displays approximately the 95th percentile of all correlation coefficients obtained by the profile comparison. Correlation coefficients above these values are considered as indicating a match between two treatments. The treatment correlation is conducted at a p-value of 0.1.

Examinations:

The metabolome as investigated in this study refers to 272 endogenous metabolites in plasma. The profiling of the metabolome was performed by means of GC-MS and LC-MS/MS. The toxicological interpretation of the metabolome data is based on the information contained in the database MetaMap® Tox at the time of the metabolome analysis. Interpretation is based on biomarkers, specific metabolite patterns, and a (statistical) comparison of the plasma metabolome changes generated by the test substance with those of other compounds present in MetaMap®Tox (currently > 900 compounds).

Details on results:

2-Propylheptanol induced significant metabolome changes at the respective dose levels. At 500 mg/kg bw/d, between 30.5-36.3% (males) and 29.4-27.9% (females) of metabolites were significantly changed, whereas at 200 mg/kg bw/d between 8.7-25.5% (males) and 10-20.8% (females) of the metabolites differed significantly from the control still lying significantly above the false discovery rate of 5%. The changed metabolites in male animals were mostly fatty acids and complex lipids such as phosphatidylcholines, whereas in female animals mostly triacylglyceride levels were increased going along with decreased amino acid and central energy metabolism intermediate levels. This indicates that the treatment with 2-Propylheptanol leads to significantly altered lipid metabolism, which is accompanied by a changed central carbon metabolism in female animals as well.
When compared to the metabolite patterns for certain toxicological endpoints available in MetaMap®Tox database the liver in relation with peroxisome proliferation was the main target organ in male animals treated with 500 mg/kg bw/d 2-Propylheptanol. In female animals, weak matches for modulated pancreas activity as well as for tubular necrosis in the kidneys were obtained. Peroxisome proliferation in the liver (male animals) and the weak match with the pattern for the modulation of pancreas activity (female animals) were also found for the low dose treatment with 2-Propylheptanol. The pattern for modulated pancreas activity is based on reference substances inducing an enhanced glucose uptake in the liver. This leads to lower amino acid plasma levels as a secondary effect, which might be also caused by peroxisome proliferators due to the changes in lipid metabolism leading to a higher availability of fatty acids for β-oxidation. Many other peroxisome proliferators, such as fibrates and phthalates induce weak matches for the respective pattern.
The correlation analysis of the whole plasma metabolite profile of male animals treated with 2-Propylheptanol with all other treatments available in the MetaMap®Tox database did result in good correlation with many phthalates, fibrates and other treatments, which induce peroxisome proliferation in the liver. In female animals the correlation analysis did not yield a conclusive result. A few phthalates were found above the threshold but not to such an extent as in male animals. The correlation analysis for the low dose treatment with 2-Propylheptanol in male animals showed a good correlation with some peroxisome proliferators and structurally related alcohols, such as 2-Ethylhexanol. In female animals, 2 out of 4 treatments above the threshold were peroxisome proliferators, whereas one was the respective high dose of 2-Propylheptanol itself.

Bis-(2-propylheptyl) phthalate induced significant metabolome changes at the respective dose levels. At 15000 / 7500 ppm, between 27.2-29.4% (males) and 29.8-34.1% (females) of metabolites were significantly changed, whereas at 3000 ppm between 16.3-34.8% (males) and 14-18.9% (females) of the metabolites differed significantly from the control still lying significantly above the false discovery rate of 5%. The changed metabolites in male animals were mostly lipid, with reduced fatty acid levels and increased sphingomyelin levels. This reflects peroxisome proliferation in the liver, which leads to an increased degradation of long-chain fatty acids, whereas activation of peroxisome proliferator-activated receptor (PPAR) γ2 also leads to de novo synthesis of sphingomyelins (Bentley et al. 1993; Li et al. 2013). Androstendione and Testosterone plasma levels were increased as well, which can be found for several peroxisome proliferation inducing treatments, which might be due to the stimulatory influence of testosterone on PPARα in the liver (Jalouli et al. 2003). In female animals mostly triacylglyceride levels were increased going along with decreased amino acid levels. This indicates that the treatment with Bis-(2-propylheptyl) phthalate leads to significantly altered lipid metabolism, which is accompanied by a changed central carbon metabolism in female animals as well.
When compared to the metabolite patterns for certain toxicological endpoints available in MetaMap®Tox database the liver in relation with peroxisome proliferation was the main target organ in male animals treated with 15000 / 7500 ppm Bis-(2-propylheptyl) phthalate. In female animals the liver related to peroxisome proliferation was the major target. In addition, weak matches for a diuretic effect on the kidney as well as a suppression of the bone marrow were found.
Peroxisome proliferation in the liver was also found for the low dose treatment with Bis-(2-propylheptyl) phthalate in male animals. In female animals, pattern for estrogenic substances were found. However, these patterns are at a preliminary state, do not contain any hormones themselves and are currently under re-evaluation to include these. The metabolites covered in these patterns consist mostly of lipids (Annex 5), which might be changed as well due to peroxisome proliferation. Therefore, these findings should not be over-interpreted and are not judged to be biologically relevant.
The correlation analysis of the whole plasma metabolite profile of male and female animals treated with Bis-(2-propylheptyl) phthalate with all other treatments available in the MetaMap®Tox database did result in good correlation with many phthalates, phenoxy herbicides, fibrates and other treatments, such as Wy14643, which induce peroxisome proliferation in the liver. The metabolic profile obtained with the low dose treatment of Bis-(2-propylheptyl) phthalate in male animals showed a good correlation with different peroxisome proliferators as well. In female animals, the correlation analysis did not yield a fully conclusive result. However, it is well-known that female rats are less sensitive to peroxisome proliferators as PPAR α is less expressed in female as compared to male liver (Jalouli et al., 2003). Norethindrone acetate was the best match, which mainly results from their common changes in lipid metabolism (Annex 6).

2-Propylheptanol and Bis-(2-propylheptyl) phthalate have been compared with each other in this study to clarify if Bis-(2-propylheptyl) phthalate is a valid source substance for a read across to 2-Propylheptanol. Both treatments have the liver as their major target organ leading to peroxisome proliferation. Thereby, Bis-(2-propylheptyl) phthalate seems to be a stronger inducer, since at roughly equivalent doses, it had also an effect on female animals. Female animals are well-known to be less sensitive to peroxisome proliferation, since PPARα is expressed to a significant lower amount in the liver of female animals (Jalouli et al. 2003). In the treatment correlation analysis of 2-Propylheptanol with all other treatments available in the MetaMap®Tox database, Bis-(2-propylheptyl) phthalate was found in the top ranks for male animals, together with other peroxisome proliferators supporting that this is the main effect of both treatments. In female animals, however, this was not the case. Therefore, a principal component analysis was conducted with both treatments and the structurally similar 2 Ethylhexanol, which was also ranked high in the treatment correlation of male animals treated with 2-Propylheptanol. Here, a clear separation of both treatments from 2 Ethylhexanol was observed for both sexes, whereas 2-Propylheptanol and Bis-(2-propylheptyl) phthalate clustered together. To investigate where this discrepancy between the treatment correlation in female animals and the principal component analysis is coming from the metabolite changes themselves were compared in detail. It was shown, that although sometimes not significant, the metabolites changed in one of the treatments are to a very large extent also changed in the same direction in the other treatment as well (Annex 7). The algorithms in the MetaMap®Tox database (Pattern Ranking and treatment correlation) are however mostly driven by significance, which is not the case for the principal component analysis. Therefore, although at some points not statistically significant, the metabolic changes induced by both treatments are very similar.

Conclusions:

Based on the obtained metabolic changes induced by the treatment with 2-Propylheptanol and Bis-(2-propylheptyl) phthalate the liver was identified as the major target organ. The strong and conclusive changes in lipid metabolism are indicative for peroxisome proliferation in the liver. The sole statistical significance of the respective metabolite changes in females were not clearly supporting similarity, most likely this may reflect the reduced sensitivity of females to PP as outlined above. However, the principal component analysis and the direct comparison of metabolic changes lead to the conclusion, that the treatment with 2-Propylheptanol will lead to the same effects as Bis-(2-propylheptyl) phthalate and accordingly, the read-across from source substance 2 to the target substance is justified. Therefore, it is concluded, that the treatment with 2-Propylheptanol will lead to the same systemic toxicological findings as Bis-(2-propylheptyl) phthalate.

Description of key information

Based on the obtained metabolic changes induced by the treatment with 2-Propylheptanol and Bis-(2-propylheptyl) phthalate the liver was identified as the major target organ. The strong and conclusive changes in lipid metabolism are indicative for peroxisome proliferation in the liver. The sole statistical significance of the respective metabolite changes in females were not clearly supporting similarity, most likely this may reflect the reduced sensitivity of females to PP. However, the principal component analysis and the direct comparison of metabolic changes lead to the conclusion, that the treatment with 2-Propylheptanol will lead to the same effects as Bis-(2-propylheptyl) phthalate and accordingly, the read-across from source substance 2 to the target substance is justified. Therefore, it is concluded, that the treatment with 2-Propylheptanol will lead to the same systemic toxicological findings as Bis-(2-propylheptyl) phthalate.