Bis(2-ethylhexyl) malate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/12/2017 to 23/3/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) malate

EC Number:

260-070-5

EC Name:

Bis(2-ethylhexyl) malate

Cas Number:

56235-92-8

Molecular formula:

C20H38O5

IUPAC Name:

1,4-bis(2-ethylhexyl) 2-hydroxybutanedioate

Test material form:

liquid

Details on test material:

Identification: Dermol DOM
Appearance: Clear colourless to pale yellow liquid
Batch: P7677
Purity/Composition: >95%
Test item storage: At room temperature
Stable under storage conditions until: 31 December 2018 (retest date)
Additional information
Test Facility test item number: 209081/A
Purity/Composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Molecular structure: Not indicated
Molecular formula: C20H38O5
Molecular weight: 358.5

Specific details on test material used for the study:

Dermol DOM also known as Diethylhexyl Malate and Dioctyl Malate (CAS number 56235-92-8, EC Number 260-070-5), batch number P7677, was a clear colourless liquid. It was received on 31 October 2017 and stored at 15-25°C protected from light. Purity was stated as >95% and the retest date was given as 26 August 2018 (2 years from the date of manufacture), see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

3.2 Formulations Analysis
In accordance with the regulatory test guidelines applicable for this study (see Section 2.4), no analyses of the stability of the test article in administered formulations or dilutions was undertaken as fresh preparation of test article were employed.
Following discussions with the Sponsor, analyses for achieved concentration and homogeneity (where appropriate) of test article formulations were not conducted as part of this study, as this is not a requirement of the regulatory test guidelines.
The absence of such analyses is noted in the Study Director’s statement of GLP compliance.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Preliminary solubility data indicated that Dermol DOM was soluble in anhydrous analytical grade dimethyl sulphoxide (DMSO) at concentrations equivalent to at least 100 mg/mL. A maximum concentration of 5000 µg/plate was selected for Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). For Experiment 2 the maximum test concentration of 5000 µg/plate was retained for all strains in the presence of S-9. For all strains in the absence of S-9, the maximum test concentration was reduced to 2000 µg/plate based on precipitation observed in Experiment 1.
Test article stock solutions were prepared by formulating Dermol DOM under subdued lighting in DMSO with the aid of vortex mixing, to give the maximum required treatment concentration. Subsequent dilutions were made using DMSO. The test article solutions were protected from light and used within approximately 5.5 hours of initial formulation. The following concentrations were tested:
Experiment S-9 Concentration of Treatment Solution (mg/mL) Final Concentration (µg/plate)
Mutation Experiment 1 - and + 0.05 5
0.016 16
0.5 50
1.6 160
5 500
16 1600
50 5000
Mutation Experiment 2 - 0.08192 8.192
0.2048 20.48
0.512 51.2
1.28 128
3.2 320
8 800
20 2000
Mutation Experiment 2 + 0.4096 20.48
1.024 51.2
2.56 128
6.4 320
16 800
40 2000
100 5000

0.1 mL volume additions of test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions were used for all pre-incubation treatments.

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Controls
Vehicle controls comprised treatments with the vehicle DMSO and were performed using the same addition volumes per plate as the test article treatments, 0.1 mL or 0.05 mL for pre-incubation treatments. Positive control treatments comprised treatments with the appropriate positive control chemicals using 0.05 mL additions per plate. The positive control chemicals were supplied and used according to the following table:
Chemical a Stock b Concentration (µg/mL) Final Concentration (µg/plate) Strain(s) S -9
2-nitrofluorene (2NF) 100 5 TA98 -
Sodium azide (NaN3) 40 2 TA100, TA1535 -
9-aminoacridine (AAC) 1000 50 TA1537 -
Mitomycin C (MMC) 4 0.2 TA102 -
Benzo[a]pyrene (B[a]P) 200 10 TA98 +
2-aminoanthracene (AAN) 100 5 TA100, TA1535, TA1537 +
400 20 TA102 +

a Obtained from Sigma-Aldrich.
b Stock solutions were formulated in purified water (NaN3 and MMC), or in DMSO (2NF, AAC, AAN and B[a]P). All stock solutions were stored in aliquots protected from light at 2-8°C, with the exception of B[a]P which was stored in aliquots at <-50ºC and MMC which was prepared freshly on the day of use or stored in aliquots at -50ºC.

Metabolic Activation System
The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P 450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities). See Quality Control Statement for S-9.
Treatments were carried out both in the absence and presence of S-9 by addition of either buffer solution or 10% S-9 mix respectively. The composition of the MutazymeTM 10% S-9 mix and buffer solution are described in the following table:
Ingredient Final Content per mL in:
10% S-9 mix Buffer Solution
Sodium phosphate buffer pH 7.4 (SPB) 100 µmoles 100 µmoles
Glucose-6-phosphate (disodium) (G-6-P) 5 µmoles -
-Nicotinamide adenine dinucleotide phosphate (NADP) (disodium) 4 µmoles -
Magnesium chloride (MgCl2) 8 µmoles -
Potassium chloride (KCl) 33 µmoles -
Water To volume To volume
S-9 100 µL -

Supplements
L-histidine HCl (in 250 mM MgCl2) and D-biotin were added at the time of plating by supplementing the molten top agar. Quantities of each supplement were as follows:
Supplement Final Quantity
L-histidine HCl 20 µg
D-biotin 24.4 µg

Bacteria
Five strains of Salmonella typhimurium bacteria (TA98, TA100, TA1535, TA1537 and TA102) were used in this study. Strains TA98, TA1535 and TA1537 were originally obtained from the UK NCTC. Strains TA100 and TA102 were derived from cultures originally obtained from Covance Laboratories Inc., USA. For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate, to provide bacterial cultures in the range of approximately 108 to 109 cells/mL, based on cell count data from testing of each strain batch. Incubation was carried out with shaking in an anhydric incubator, set to turn on using a timer switch. All treatments were completed within 6 hours of the end of the incubation period.
The inocula were taken from master plates or vials of frozen cultures, which had been checked for strain characteristics (histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline).

Evaluation criteria:

For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For 5,000 mg/l experiment only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For 5,000 mg/l experiment only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For 5,000 mg/l experiment only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

For 5,000 mg/l experiment only

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Results

Toxicity, Solubility and Concentration Selection

Details of all treatment solution concentrations and finalDermol DOMconcentrations are provided in the Test Article Section3.1.

Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations ofDermol DOMat 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity in the form of a thinning of the background bacterial lawn was observed in the presence of S-9 only, and occurred at 5000 µg/plate in all strains, except TA98. Precipitation of test article was observed on all plates treated at 500 µg/plate and above in the absence of S-9, and at 1600 µg/plate and above in all strains except TA98 in the presence of S-9, where precipitation was only observed at 5000 µg/plate.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for treatments of all strains in the presence of S-9. For all strains in the absence of S‑9, the maximum test concentration was reduced to 2000 µg/plate based on precipitation observed in Experiment 1. Narrowed concentration intervals were employed covering the ranges 20.48-5000 µg/plate in the presence of S-9 or 8.192‑2000 µg/plate in the absence of S-9, in order to examine more closely those concentrations ofDermol DOMapproaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity in the form of thinning of the background bacterial lawn was limited to the 5000 µg/plate treatments of strain TA102 in the presence of S-9. Precipitation of test article was observed on all plates treated at 2000 µg/plate and above.

Data Acceptability and Validity

The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section8.From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges(Section7.3). The positive control chemicals all induced increases in revertant numbers of≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3‑fold (in strains TA1535 and TA1537) the concurrent vehicle controlsconfirming discrimination between different strains, and an active S‑9 preparation. It was noted that although a>3‑fold increase was observed with the positive control treatments in strain TA1537 in the presence of S-9 in Experiment 2, the revertant numbers were slightly below the laboratory historical control range for these positive control treatments. However, as these positive control treatments provided a greater than 3-fold increase over the concurrent vehicle control level, and as the correct strain functioning was also confirmed by the control data in this strain in the absence of S-9, and the metabolic activity of the S-9 mix also confirmed by the positive control responses in the other tester strains in the presence of S-9, these data were considered both acceptable and valid.The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation

FollowingDermol DOMtreatments of all the test strains in the absence and presence of S-9, no notable and concentration-related increases in revertant numbers were observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of anyDermol DOMmutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:

It was concluded that Dermol DOM did not induce mutation in five histidine requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (a precipitating concentration and the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

Dermol DOMwas assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

AllDermol DOMtreatments in this study were performed using formulations prepared in anhydrous analytical grade dimethyl sulphoxide (DMSO).

Experiment 1 treatments of all thetester strains were performed in the absence and in the presence of S-9, using final concentrations ofDermol DOMat 5, 16, 50, 160, 500, 1600and 5000 µg/plate. Following these treatments, evidence of toxicity was observed in the presence of S-9 only, and occurred at 5000 µg/plate in all strains, except TA98. Precipitation of test article was observed on all plates treated at 500 µg/plate and above in the absence of S-9, and in the presence of S-9 at 5000 µg/plate in all strains and also at 1600 µg/plate in all strains except TA98.

Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for the treatments of all strains in the presence of S-9.For all strains in the absence of S-9, the maximum test concentration was reduced to 2000 µg/plate based on precipitationobserved in Experiment 1. Narrowed concentration intervals were employed covering the ranges 20.48-5000 µg/plate in the presence of S-9 or 8.192‑2000 µg/plate in the absence of S-9, in order to examine more closely those concentrations ofDermol DOMapproaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, evidence of toxicity was limited to the 5000 µg/plate treatments of strain TA102 in the presence of S-9. Precipitation of test article was observed on all plates treated at 2000 µg/plate and above.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell withinacceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

FollowingDermol DOMtreatments of all the test strains in the absence and presence of S-9, no notable and concentration-related increases in revertant numbers were observed, and none that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of anyDermol DOMmutagenic activity in this assay system.

It was concluded thatDermol DOMdid not induce mutation in five histidine‑requiring strains (TA98, TA100, TA1535, TA1537 and TA102) ofSalmonella typhimuriumwhen tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (a precipitating concentration and the maximum recommended concentration according to current regulatory guidelines) in the absence and in the presence of a rat liver metabolic activation system (S-9).