Propyl acetate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Endpoint:

mechanistic studies

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The analysis 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 report preparation. 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).

GLP compliance:

no

Remarks:

Investigations were carried out in accordance with the Standard Operating Procedures (SOPs)

Type of method:

other: Metabolome study

Endpoint addressed:

neurotoxicity

immunotoxicity

other: endocrine mode of action

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: BASF SE, Ludwigshafen, Germany
- Batch No.of test material:
83294456P0
- Purity:
99.915%
- Expiry date: 10 Dec 2017
- Identity: confirmed
- Physical state/appearance: Liquid/colorless, clear

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room temperature
- Stability under storage conditions:
Stability of the test substance under storage conditions over the test period was guaranteed.
- Homogeneity: given (visually)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The specific amount of test substance was weighed, topped up with corn oil in a graduated flask and intensely mixed by magnetic stirrer until it dissolved.

Species:

rat

Strain:

Wistar

Remarks:

The rat is the preferred animals species for developmental and reproductive toxicity studies according to the various test guidelines. This Wistar rat strain (Crl:WI(Han)) was selected because extensive historical control data is available for these rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 13 weeks
- Weight at study initiation: not specified
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6 am to 6 pm illumination)

IN-LIFE DATES: From: 15 Feb 2017 To: 07 Mar 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Volume to be administered: 4 ml/kg bw

DIET PREPARATION
- Rate of preparation of diet (frequency): at intervals
- Mixing appropriate amounts with: Amount of test substance was weighed, topped up with corn oil and intensely mixed
- Storage temperature of preparation: room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

daily

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

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 15 from overnight fasted animals under isoflurane anesthesia and the obtained EDTA-plasma was covered with nitrogen and frozen at -80°C.

Examinations:

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 chromatography-mass 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 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 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).

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.

Positive control:

no positive control included

Details on results:

No treatment-related clinical signs or mortality were observed. Body weight and body weight changes were not affected. At 1000 mg/kg bw/day, female animals displayed significantly decreased food consumption (-10.6%) which was, however, not reflected in the body weight. Water consumption, clinical chemistry, ophthalmological examinations, histopathology, and gross pathology were not examined.

CHANGED METABOLITES - ASSESSMENT OF KEY CHANGES

- At a dose level of 1000 mg/kg bw/day, n-propyl acetate induced no changes in the metabolite profile compared to the control (no. of significantly changed metabolites below the false positive rate at p < 0.05 of 14 metabolites; based on 274 possible metabolites). The profile strength is in good accordance with the number of significantly changed metabolites, indicating no effect of n-Propyl acetate on the plasma metabolome of rats of both sexes.
- In male animals Dodecanol, 16-Methylheptadecanoic acid, sucrose, serine and an unknown polar were significantly downregulated, whereas panthothenic acid, pyruvate and 3,4-dihydroxyphenylglycol were significantly upregulated. In female animals cholic acid, glucose, lysophosphatidylcholine (C20:4) as well as the two amino acids valine and glutamate were significantly downregulated. 3,4-dihydroxyphenylalanine, on the other hand, was significantly upregulated.

- At 300 mg/kg bw/day, the number of metabolite changes induced by n-Propyl acetate lay below the false positive rate (14 metabolites based on 274 possible metabolites at p < 0.05).
- In male animals, fructose-6-phosphate, phosphatidylcholine (C18:0, C20:3) as well as the two ketobodies 3-hydroxyisobutyrate and 2-hydroxybutyrate were significantly down-regulated. Creatinine and phosphocreatinine levels were significantly upregulated. In female animals the amino acids cytosine, histidine and isoleucine as well as deoxyribonucleic acids, 3-phosphoglycerate, 3- methoxytyrosine and different hexoses were significantly downregulated.

DETECTION OF TOXICOLOGICAL MODES OF ACTION

Pattern ranking
- At 300 and 1000 mg/kg bw/day, the comparison of the metabolite changes induced by n-Propyl acetate treatment against the established specific metabolite patterns present in MetaMap® Tox resulted in no matches or weak matches in target organs: pancreas (endocrine system), microbiome, endocrine system, bone, ovaries (endocrine system), thyroid, liver, nervous system, kidney

Profile comparison with reference compounds
- 1000 mg/kg bw/day: Using total profile comparison, the metabolite profile of 1000 mg/kg bw/day n-Propyl acetate in male and female animals resulted in only one match, namely its own low dose.
- 300 mg/kg bw/day: The total profile comparison for the metabolite profile of 300 mg/kg bw/day n-Propyl acetate in male and female animals did match only with its own high dose

Table1:Metabolite changes in the300and1000 mg/kg bw/da ydose groups ofn-Propyl acetate

	n-Propyl acetate
	14 days
	300 mg/kg bw/day		1000 mg/kg bw/day
	females	males	females	males
Number down regulated	7	4	6	5
Number Up regulated	0	2	1	3
Number total	7	6	7	8
% down regulated	2.8	1.6	2.4	2.0
% up regulated	0.0	0.8	0.4	1.2
% total	2.8	2.4	2.8	3.2
Profile Strength	0.8	0.67	0.89	0.83

Conclusions:

Even at the limit dose of 1000 mg/kg bw/day, the induced metabolite changes were below the false discovery rate. No signs of systemic toxicity were found after 14 days of treatment with n-Propyl acetate in male and female Wistar rats and no hints for the presence of triggers (with regard to an endocrine mode of action, neurotoxicity and/or immunotoxicity) for the different cohorts of the extended one-generation reproductive toxicity study (EOGRTS according to OECD 443 guideline) could be identified.

Executive summary:

This report contains information on the metabolite profile (metabolome) in plasma of rats treated with n-Propyl acetateby gavage for 14 days. Five animals per sex per dose were treated with 300 and 1000 mg/kg bw/day orally by gavage for the study period.Blood samples for metabolome analysis were taken by puncturing the retrobulbar venous plexus on study day15from overnight fasted animals under isoflurane anesthesia.The plasma metabolome was examined using GC-MS and LC-MS/MS techniques.The sex- and day-stratifiedheteroscedastic t-test("Welch test") was applied to compare metabolite levels of dose groups with respective controls.n-Propyl acetateinduced only minimal metabolome changes even below the false discovery rate (p = 0.05) at both dose levels. None of the found metabolite changes were detected in a dose-dependent manner or even at both dose levels, which strongly indicates that these changes are probably not treatment related. No statistically significant changes were found for sex hormones or related substances. Only statistically significant changes (p = 0.05) could be interpreted as biologically relevant.Based on the here obtained findings there is no specific indication for a possible endocrine mode of action or an influence on steroid metabolism.Neither the high dose treatment nor the low dose treatment withn-Propyl acetateresulted in any matches or weak matches when compared to the metabolite patterns for certain toxicological modes of action available in the MetaMap^®Tox database, thus no trigger for any of the additional cohorts of the EOGRTS could be identified. This can also be explained by the very few changes induced by the treatment withn-Propyl acetateeven at the limit dose of 1000 mg/kg bw/day.It is therefore not to be expected that the treatment withn-Propyl acetateleads to any of the here covered modes of action.The correlation analysis of the whole plasma metabolite profile of animals treated withn-Propyl acetate with all other treatments available in the MetaMap^®Tox database did also not hint at an endocrine mode of action or a potential for induction of neurotoxicity or immunotoxicity of the test substance. In all cases only the respective high dose or low dose treatment was found above the threshold of 0.4. for males and 0.5 for females and no treatment present in the database correlated well with n-Propyl acetate.

Description of key information

Five Wistar rats /sex/dose treated with 300 and 1000 mg/kg bw/day. Blood was taken for metabolome analysis. Induced metabolite changes were below the false discovery rate. No hints for the presence of triggers (with regard to an endocrine mode of action, neurotoxicity and/or immunotoxicity) could be identified (BASF SE, 2018). Non-guideline, Non-GLP.

Additional information

Metabolome study

This report contains information on the metabolite profile (metabolome) in plasma of rats treated with n-Propyl acetateby gavage for 14 days. Five animals per sex per dose were treated with 300 and 1000 mg/kg bw/day orally by gavage for the study period.Blood samples for metabolome analysis were taken by puncturing the retrobulbar venous plexus on study day15from overnight fasted animals under isoflurane anesthesia.The plasma metabolome was examined using GC-MS and LC-MS/MS techniques.The sex- and day-stratifiedheteroscedastic t-test("Welch test") was applied to compare metabolite levels of dose groups with respective controls.n-Propyl acetateinduced only minimal metabolome changes even below the false discovery rate (p = 0.05) at both dose levels. None of the found metabolite changes were detected in a dose-dependent manner or even at both dose levels, which strongly indicates that these changes are probably not treatment related. No statistically significant changes were found for sex hormones or related substances. Only statistically significant changes (p = 0.05) could be interpreted as biologically relevant.Based on the here obtained findings there is no specific indication for a possible endocrine mode of action or an influence on steroid metabolism.Neither the high dose treatment nor the low dose treatment withn-Propyl acetateresulted in any matches or weak matches when compared to the metabolite patterns for certain toxicological modes of action available in the MetaMap^®Tox database, thus no trigger for any of the additional cohorts of the EOGRTS could be identified. This can also be explained by the very few changes induced by the treatment withn-Propyl acetateeven at the limit dose of 1000 mg/kg bw/day.It is therefore not to be expected that the treatment withn-Propyl acetateleads to any of the here covered modes of action.The correlation analysis of the whole plasma metabolite profile of animals treated withn-Propyl acetate with all other treatments available in the MetaMap^®Tox database did also not hint at an endocrine mode of action or a potential for induction of neurotoxicity or immunotoxicity of the test substance. In all cases only the respective high dose or low dose treatment was found above the threshold of 0.4. for males and 0.5 for females and no treatment present in the database correlated well with n-Propyl acetate.