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Reference
Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2012-2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
Plasma was taken from fasted rats after 28-day treatment during the course of the 90day study (OECD 408, GLP; mid and high dose group). The plasma metabolome, i.e. 225 endogenous plasma components such as amino acids, hormones, sugar, fatty acids, etc., was investigated in regard to changes relative to the control group. 72 of the of 225 components were determined in by two independent methods so that the overall number of measurements is 297.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
other: Changes in the plasma metabolome consisting of 225 endogenous plasma components
Specific details on test material used for the study:
- Name of test material (as cited in study report): Reaction products of benzeneamine, N-phenyl with nonene (branched)
- Substance type: viscous, slightly yellow
- Physical state: liquid
- Analytical purity: UVCB-substance, purity not applicable
- Purity test date: 2013
- Lot/batch No.: 240312/K7 Lot. Nr. 020
- Expiration date of the lot/batch: 19 Feb 2014
- Stability under test conditions: stable
- Storage condition of test material: Ambient (room temperature); avoiding temperatures <10 °C
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
See IUCLID Chapter 7.5.1 for the 90-day-study with CAS 36878-20-3.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
See IUCLID Chapter 7.5.1 for the 90-day-study with CAS 36878-20-3.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Post exposure period:
none
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
Blood samples for metabolome analyis were taken retroorbitally on study day 29 from overnight fasted animals under isoflurane anesthesia. The obtained EDTA-plasma was covered with nitrogen and frozen at -80°C.

Sample work up involved GC/MS and LC/MS-MS techniques.
The plasma metabolome was examined by metanomics GmbH following proprietary sample work up using GC/MS and LC/MS-MS techniques. Briefly, three types of mass spectrometry analysis were applied to all samples: GC-MS (gas chromatographymass spectrometry) and LC-MS/MS (liquid chromatography-MS/MS) were used for broad profiling, as described in van Ravenzwaay et al. (2007). SPE-LC-MS/MS (Solid phase extraction-LC-MS/MS) was applied for the determination of catecholamine and steroid hormone levels. Proteins were removed from plasma samples by precipitation.
Subsequently polar and non-polar fractions were separated for both GC-MS and LCMS/ 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 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). Steroids hormones, catecholamines and their metabolites were measured by online SPE-LC-MS/MS (Solid phase extraction- LC-MS/MS) (Yamada et al., 2002). 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).




297 molecules were determined.
Examinations:
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 297 analytes, 15 significant metabolite changes can be expected on a significance level of 0.05 (“false positive” rate). Therefore, up to 15 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
2) Using an established algorithm, the similarity of the test compound metabolic profile with the predefined patterns in MetaMap®Tox (> 120 patterns currently covering 42 modes of action) was determined and evaluated by an expert panel.

The outcome of this assessment is one of four defined categories: The metabolite changes match a certain mode of action (“match”), the metabolic change is weakly associated with a mode of action (“weak match”), no conclusion is possible (“equivocal”) or the metabolic change does not match with a mode of action (“mismatch”). 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 “profile comparison” using Spearman and Pearson correlations. Based on
the reference data base, a threshold value of 0.50 for male animals and 0.60 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 clear match between two treatments.
Details on results:
The substance did not show matches (with those of compounds present in MetaMap®Tox 1) which would give a clear indication for a certain toxicological mode of action.
At 300 and 1000 mg/kg bw, a significant and dose-dependent effect on the number of metabolites was observed. The dose group of 100 mg/kg bw was not examined. Females showed more changes than males.
From the affected endogenous substances, it appears that the substance affects lipid metabolism and has diffuse effects on liver. At the high dose group, the reduced body weight has an impact on the pattern.

The changes were compared to those obtained after 28-day treatment with CAS 68411-46-1. It was found that a majority of metabolome changes was similar for both compounds in terms of significance and direction of change (either increased or decreased.) Applying a Pearson-based statistical correlation of the whole plasma metabolome, CAS 36878-20-3 and CAS 68411-46-1 were the most similar compounds in terms of metabolome changes out of a data base consisting of more than 750 substances.

Description of key information

The effects of treatment on 297 plasma parameters after 28-day treatment was investigated. Changed parameters are indicative of effects on liver and a disturbance of lipid metabolism. A similar pattern of change was observed for CAS 36878 -20 -3 and CAS 68411 -46 -1.

Additional information