Reaction mass of pentyl ester butanoic acid and 2-methylbutyl ester butanoic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies presented in this section were conducted in accordance to recognised testing guidelines and with GLP certification.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 April 2019 - 17 June 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

Methods for the Determination of Toxicity and other health effects, 31 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name: Reaction mass of 2-methylbutyl butyrate and pentyl butyrate
EC number: 908-712-1
Description: Clear colorless to pale yellow liquid
Purity: 99.8%
Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions.

The Salmonella typhimurium strains are constructed to differentiate between base-pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.

The Escherichia coli WP2 uvrA tryptophan (trp) reversion system measures trp- → trp+ reversions.

The Escherichia coli WP2 uvrA detects mutagens that cause base-pair substitutions (AT to GC)

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Mammalian microsomal enzyme activation mixture (liver extract, S9 fraction).

The activation system uses nicotinamide-adenine dinucleotide phosphate (NADP+)-cytochrome P450 dependent mixed function oxidase enzymes of the liver.
The liver extract was obtained from rats, which were pre-treated with phenobarbital and β-naphthoflavone, two inducers of several drug-metabolizing enzymes.

METABOLIC ACTIVATION SYSTEM
Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
 
The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of Citoxlab Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
The composition of solution refers to a final volume of 1000 mL.
 
INDUCTION OF LIVER ENZYMES
Male Wistar rats (349-395 g, animals were 9-10 weeks old at initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Euthanasia was performed by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used for preparation S9 used in this study was 27 November 2018.
 
PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
 
On Day 4, the rats were euthanized and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10 ºC. The date of preparation of S9 fraction for this study was 30 November 2018 (Citoxlab code: E13013, Expiry date: 30 November 2020).
The sterility of the preparation was confirmed in each case. The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of Citoxlab Hungary Ltd. The mean protein concentration of the S9 fraction used was determined to be 26.7 g/L.
The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. Each batch of S9 used in this study functioned appropriately.

THE S9 MIX (containing 10 % (v/v) of s9)
 
Salt solution for S9 Mix: 
NADP Na: 7.66 g
D-glucose-6 phosphate Na: 3.53 g
MgCl2x 6 H2O: 4.07 g
KCl: 6.15 g
Distilled water: *q.s. ad 1000 mL
Sterilization was performed by filtration through a 0.22 μm membrane filter.
 
The complete S9 mix was freshly prepared containing components as follows:
Ice cold 0.2 M sodium phosphate buffer, pH 7.4: 500 mL
Rat liver homogenate (S9): 100 mL
Salt solution for S9 Mix (see above): 400 mL
Prior to addition to the culture medium the S9 mix was kept in an ice bath.
 
0.2 M SODIUM PHOSPHATE BUFFER, PH 7.4
Solution A:
Na2HPO4x 12 H2O: 71.63 g
Distilled water: *q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
 
Solution B:
NaH2PO4x H2O: 27.6 g
Distilled water: *q.s. ad 1000 mL
Sterilization was performed at 121°C in an autoclave.
 
0.2M Sodium phosphate buffer pH 7.4:
Solution A: 880 mL
Solution B: 120 mL

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In Assay 1, Assay 2 the same concentrations were used. In Assay 3 different concentrations were used because of the observed cytotoxicity in Assay 2.

Preliminary Solubility Test - At the concentration level of 100 mg/mL, insolubility was detected using Distilled water.

Preliminary Range Finding Test - 100 mg/mL stock solution was prepared in DMSO, which was diluted to 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation.

Assay 1, Assay 2 and Assay 3 - The maximum test concentration was 5000 μg test item/plate in Assay 1 and Assay 2. The maximum test concentration was 500 μg test item/plate in Assay 3.

Vehicle / solvent:

Based on the available information and the solubility and compatibility test, 100 mg/mL stock solution was prepared in DMSO.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:

- Number of cultures per concentration: triplicate
- Number of independent experiments: 3
assay 1 plate incorporation method,
assay 2 and 3 pre-incubation method,

METHOD OF TREATMENT/EXPOSURE
At the same concentration level, good solubility was detected using DMSO and DMF. Due to the better biocompatibility, DMSO was selected as vehicle for the study.100 mg/mL stock solution was prepared in DMSO.

TREATMENT AND HARVEST SCHEDULE:

Procedure for Exposure in Assay 1 - Assay 1 followed the standard plate incorporation procedure. Bacteria (cultured in Nutrient Broth No .2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system.

Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per test item concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).

The content of the tubes:
- top agar 2000 µL
- vehicle or test item formulation (or reference controls) 50 µL
- overnight culture of test strain 100 µL
- phosphate buffer (pH 7.4) or S9 mix 500 µL

This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions,
with the addition of untreated, negative (solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 hours.


Procedure for Exposure in Assay 2 and Assay 3 - Assay 2 and Assay 3 followed the standard pre-incubation procedure since in Assay 1 no positive effect was observed.

For the pre-incubation method, bacteria (cultured in Nutrient Broth No. 2) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C.

Before the overlaying, 50 µL of the test item formulation, vehicle, positive reference controls or their solvents, 100 µL of the overnight culture of bacterial cells and 0.5 mL of the S9 mix (activated test conditions) or phosphate buffer pH 7.4 (non-activated test conditions) were added into appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 minutes at 37ºC in a shaking incubator.

After the incubation period, 2 mL of molten top agar were added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 hours in Assay 2. In Assay 3 the incubation period was extended for 66 hours and 24 minutes.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Cell count (manual)

Evaluation criteria:

For evaluation criteria see 'any other information on materials and methods' section.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Using the pre-incubation method, excessive cytotoxicity was observed without metabolic activation at four concentrations; repeated using a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Using the pre-incubation method, excessive cytotoxicity was observed without metabolic activation at four concentrations; repeated using a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Using the pre-incubation method, excessive cytotoxicity was observed without metabolic activation at four concentrations; repeated using a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Using the pre-incubation method, excessive cytotoxicity was observed without metabolic activation at four concentrations; repeated using a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Using the pre-incubation method, excessive cytotoxicity was observed without metabolic activation at four concentrations; repeated using a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY COMPATIBILITY TEST

Based on the available information and the results of the solubility testing, DMSO was selected as vehicle (solvent) of the study. The results of the Preliminary Compatibility Test are summarized in Table 2 in the 'any other infromation on results' section.

PRELIMINARY RANGE FINDING TEST

In the Preliminary Range Finding Test, the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 strains in the presence and absence of metabolic activation system (± S9 mix) with appropriate untreated, negative (solvent) and positive controls. In the test, each sample (including the controls) was tested in triplicate.
Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate were examined in the Preliminary Concentration Range Finding Test.

In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).

No precipitate was observed in the Preliminary Range Finding Test in any bacterial strains with and without metabolic activation.

Slightly reduced background lawn was detected in the Preliminary Range Finding Test in both bacterial strains with and/or without metabolic activation on the plates at the concentration levels of 5000 and 2500 µg/plate in Salmonella typhimurium TA98 strain. The same effect was observed in Salmonella typhimurium TA100 strain with and without metabolic activation on the plates at the concentration levels of 5000, 2500 and 1000 µg/plate.
The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 (Appendix 2) and in Appendix 3.

MUTAGENICITY TESTS (ASSAY 1, ASSAY 2 AND ASSAY 3)
In Assay 1, the plate incorporation method was used. In Assay 2 as well as in Assay 3, the pre-incubation method was used.
The Assays 1-2 were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (±S9 mix) with appropriate untreated, negative (solvent) and positive controls. Assay 3* was carried out using the same bacterial strains in the absence of a metabolic activation system. In the main tests, each sample (including the controls) was tested in triplicate.

*Note: In Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all examined bacterial strains without metabolic activation at four concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in all examined bacterial strains without metabolic activation was repeated in an additional experiment (Assay 3) with the same conditions using the pre-incubation method and a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

In the main tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

In Assay 1 (using plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 bacterial strain at 1.581 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.46). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In Assay 2 and Assay 3 (using the pre-incubation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 15.81 μg/plate concentration with metabolic activation (the observed mutation factor value was: MF: 1.57). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

No precipitate was observed in the main tests in all examined bacterial strains with and without metabolic activation.

Slightly reduced background lawn was detected in Assay 1 in all examined Salmonella typhimurium strains with and/or without metabolic activation on the plates at the concentration levels of 5000, 1581 and 500 µg/plate. Reduced/slightly reduced background lawn was detected in Assay 2 in all examined bacterial strains with metabolic activation on the plates at the concentration levels of 5000, 1581 and/or 500 µg/plate. The same effect was detected in Assay 3 in all examined bacterial strains without metabolic activation on the plates at the concentration levels of 500 and 58.1 µg/plate.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 (Appendix 2) and Appendix 4, 5 and 6.

VALIDITY OF THE TESTS
Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.

The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test.

At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate.

The study was considered to be valid.

Table 2: The Solubility of the Test Item in DMSO

Concentration of test item in DMSO (mg/mL)	Solubility in DMSO	Solubility in the top solution (test item formulation 50 µL + phosphate buffer 500 µL + top agar 2 mL)	Test item concentration in the test plate (µg/tube)
100	clear solution	slightly opalescent, tiny oily drops	5000
50	clear solution	slightly opalescent, tiny oily drops	2500
20	clear solution	slightly opalescent	1000
6.32	clear solution	clear solution	316

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Reaction mass of 2-methylbutyl butyrate and pentyl butyrate had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.

Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

 

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Plate Incorporation Method), Assay 1 (Plate Incorporation Method), Assay 2 (Pre-Incubation Method) and Assay 3* (Pre-Incubation Method).

*Note: In Assay 2 using the pre-incubation method, excessive cytotoxicity was observed in all examined bacterial strains without metabolic activation at four concentrations. In this case, the number of analyzable doses did not meet the recommendations of the test guidelines. Therefore, the experiment in all examined bacterial strains without metabolic activation was repeated in an additional experiment (Assay 3) with the same conditions using the pre-incubation method and a modified concentration range (500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 µg test item/plate).

Based on the results of the Preliminary Compatibility Test, the test item was dissolved in Dimethyl sulfoxide. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate were examined in the Preliminary Range Finding Test in Salmonella typhimurium TA98 and TA100 tester strains. Based on the results of the Range Finding Test, the test item concentrations in Assay 1 and Assay 2 were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate, in Assay 3 were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

 

In main tests, the number of revertant colonies did not show any biologically relevant increase compared to the solvent control. There were no dose-related trends and no indication of any treatment-related effect.

No precipitate was observed in the main tests in all examined bacterial strains with and without metabolic activation.

 

 Slightly reduced background lawn was detected in Assay 1 in all examined Salmonella typhimurium strains with and/or without metabolic activation on the plates at the concentration levels of 5000, 1581 and 500 µg/plate. Reduced/slightly reduced background lawn was detected in Assay 2 in all examined bacterial strains with metabolic activation on the plates at the concentration levels of 5000, 1581 and/or 500 µg/plate. The same effect was detected in Assay 3 in all examined bacterial strains without metabolic activation on the plates at the concentration levels of 500 and 158.1 µg/plate.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

In conclusion, the test item Reaction mass of 2-methylbutyl butyrate and pentyl butyrate had no mutagenic activity on the applied bacterial strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in vitro test for bacterial mutagenicity is validated, and the results are suitable for use as the basis for classification according to the UN GHS categories. The results of the test presented in this section indicate that the registered substance does not meet the criteria for mutagenicity in accordance with the Classification, Labelling, and Packaging (CLP) regulation (1272/2008). Further in vitro testing is recommended at the next tonnage band in accordance with the requirements of REACH (1907/2006).