Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
6/12/2017 to 15/2/2018
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS 1629579-82-3
Molecular formula C18H32N2O11
EC 818-033-1
Specific details on test material used for the study:
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Batch: 11_13_17_1
Purity/Composition: Not indicated
Test item storage: At room temperature
Stable under storage conditions until: 13 November 2019 (expiry date)

Additional information
Test Facility test item number: 207396/C
Purity/Composition correction factor: Contains 74% water
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes, maximum temperature: 40°C, maximum duration: 60 minutes
Chemical name (IUPAC, synonym or trade name: Bis-Aminopropyl Diglycol Dimaleate
CAS number: 11629579-82-3
Molecular formula: C18H32N2O11
Molecular weight: 452.46
Solubility in vehicle: Water: Highly
Stability in vehicle: Water: Stable

Method

Species / strain
Species / strain:
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:
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 0.44, 1.4, 4.4, 14, 43, 133, 416 and 1299 µg/plate were tested in triplicate.
The highest concentration of the test item used in the first and second mutation experiment was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

Bis-Aminopropyl Diglycol Dimaleate was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 0.44, 1.4, 4.4, 14, 43, 133, 416 and 1299 µg/plate in the absence and presence of S9-mix.
Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation assay with the tester strains, TA1535, TA1537 and TA98, in the absence and presence of S9-mix: 41, 129, 403, 1259, 3935 μg/plate.
Since in the dose-range finding test, no toxicity and no precipitate on the plates was observed at the highest dose level tested, the tester strains TA100 and WP2uvrA were again tested in the first mutation experiment. The test item was tested at the dose level of 3935 μg/plate in the tester strains TA100 and WP2uvrA in the absence and presence of S9-mix.
Vehicle:
The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, ICR-191 (Sigma)
Remarks:
Solvents for Positive Control Items; Saline = physiological saline DMSO = dimethyl sulfoxide
Details on test system and conditions:
Preparation of Test Item
A correction factor of 3.85 (according to the water content) was applied in this study.
A solubility test was performed based on visual assessment. The test item was dissolved in Milli-Q water.
Test item concentrations were used within 2.5 hours after preparation.
Any residual volumes were discarded.

Test System

Test System Salmonella typhimurium bacteria and Escherichia coli bacteria
Rationale Recommended test system in international guidelines (e.g. OECD, EC).
Source Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535: 2016, TA1537: 2015, TA98: 2017, TA100: 2017; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA: 2008)]

The characteristics of the different Salmonella typhimurium strains were as follows:
Strain Histidine mutation Mutation type
TA1537 hisC3076 Frameshift
TA98 hisD3052/R-factor* Frameshift
TA1535 hisG46 Base-pair substitutions
TA100 hisG46/R-factor* Base-pair substitutions
*: R-factor = plasmid pKM101 (increases error-prone DNA repair)

Each tester strain contained the following additional mutations:
rfa : deep rough (defective lipopolysaccharide cellcoat)
gal : mutation in the galactose metabolism
chl : mutation in nitrate reductase
bio : defective biotin synthesis
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene)

The Salmonella typhimurium strains were regularly checked to confirm their
histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100),
UV-sensitivity and the number of spontaneous revertants.

The Escherichia coli WP2uvrA strain detects base-pair substitutions. The strain lacks an excision repair system and is sensitive to agents such as UV. The sensitivity of the strain to a wide variety of mutagens has been enhanced by permeabilization of the strain using
Tris-EDTA treatment (Ref.1). The strain was regularly checked to confirm the
tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants.
Stock cultures of the five strains were stored in liquid nitrogen (-196°C).

Cell Culture

Preparation of bacterial cultures
Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C,
150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/mL). Freshly grown cultures of each strain were used for a test.

Agar plates
Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 µg/plate biotin (Merck) and 15 µg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 µg/plate tryptophan (Sigma).

Top agar
Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for
20 min at 121 ± 3°C.

Environmental conditions
All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 34.8 - 40.1°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System

S9-Fraction
Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and
2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 µg/plate and 2.5 µg/plate, respectively.

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 mL: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg
glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 mL Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 mL 0.5 M sodium phosphate buffer pH 7.4; 1 mL 0.08 M MgCl2 solution (Merck); 1 mL 0.33 M KCl solution (Merck). The above solution was filter (0.22 µm)-sterilized. To 9.5 mL of S9-mix components 0.5 mL S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

Positive Controls

Without Metabolic Activation
Strain Chemical Solvent Concentration/plate Concentration/plate
Direct plate assay Pre-incubation assay
TA1535 sodium azide (SA) Saline 5 µg 5 µg
TA1537 ICR-191 DMSO 2.5 µg
TA1537 2-nitrofluorene (NF) DMSO 15 µg
TA98 2-nitrofluorene (NF) DMSO 10 µg 10 µg
TA100 methylmethanesulfonate (MMS) DMSO 650 µg 650 µg
WP2uvrA 4-nitroquinoline N-oxide DMSO 10 µg 10 µg

With Metabolic Activation
Strain Chemical Solvent Concentration/plate Concentration/plate
Direct plate assay Pre-incubation assay
TA1535 2-aminoanthracene (2AA) DMSO 2.5 µg 2.5 µg
TA1537 2-aminoanthracene (2AA) DMSO 2.5 µg 2.5 µg
TA98 2-aminoanthracene (2AA) DMSO 1 µg 1 µg
TA100 2-aminoanthracene (2AA) DMSO 1 µg 5 µg
WP2uvrA 2-aminoanthracene (2AA) DMSO 15 µg 15 µg

Solvents for Positive Control Items

Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands)
DMSO = dimethyl sulfoxide (Merck, Darmstadt, Germany)

Evaluation criteria:
ACCEPTABILITY CRITERIA
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

First Experiment: Direct Plate Assay

Bis-Aminopropyl Diglycol Dimaleate was initially tested in the tester strains TA100 and WP2uvrAas a dose-range finding test with concentrations of 0.44, 1.4, 4.4, 14, 43, 133, 416 and 1299 µg/plate in the absence and presence of S9-mix.   

Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation assay with the tester strains, TA1535, TA1537 and TA98, in the absence and presence of S9-mix: 41, 129, 403, 1259, 3935 μg/plate. 

Since in the dose-range finding test, no toxicity and no precipitate on the plates was observed at the highest dose level tested, the tester strains TA100 and WP2uvrAwere again tested in the first mutation experiment. The test item was tested at the dose level of 3935 μg/plate in the tester strains TA100 and WP2uvrAin the absence and presence of S9-mix.

The results are shown inTable 1andTable2. The individual data are presented inAppendix 3.

Precipitate

Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain. 

Toxicity

To determine the toxicity of the test item, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed. The definitions are stated inAppendix 2.

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

First Experiment (additional): Direct Plate Assay

Since in the first mutation assay, no toxicity and no precipitate on the plates was observed at the highest dose level tested in all five tester strains, an additional mutation experiment was performed. In the additional experiment the test item was tested at the dose level of
5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

The results are shown inTable 1andTable2. The individual data are presented inAppendix 3.

Precipitate

Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in any tester strain. 

Toxicity

No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

Mutagenicity

In the direct plate test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Second Experiment: Pre-Incubation Assay

To obtain more information about the possible mutagenicity of the test item, a pre-incubation experiment was performed in the absence and presence of S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA

The results are shown inTable3, the individual data are presented inAppendix 3.

Precipitate

Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period. 

Toxicity

There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity

In the pre-incubation test, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Discussion

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments. 

The negative control values were within the laboratory historical control data ranges, except the response for TA98 in the absence of S9-mix in the additional first experiment. However since the mean number of revertant colonies showed a characteristic number of revertant colonies (7 revertant colonies) when compared against relevant historical control data
(8 revertant colonies), the validity of the test was considered to be not affected.

The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the results of this study it is concluded that Bis-Aminopropyl Diglycol Dimaleate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The objective of this study was to determine the potential of Bis-Aminopropyl Diglycol Dimaleate and/or its metabolites to induce reverse mutations at the histidine locus in several strains ofSalmonellatyphimurium(S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus ofEscherichiacoli(E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. An additional direct plate assay was performed to complete the data of the first experiment.

The study procedures described in this report were based on the most recent OECD and EC guidelines.

Batch 11_13_17_1 of Bis-Aminopropyl Diglycol Dimaleate was a clear to transparent yellow liquid. A correction factor of 3.85 was used to correct for the water content (74%). The test item was dissolved in water. 

In the dose-range finding study, the test item was initially tested up to concentrations of
1299 µg/plate in the strains TA100 and WP2uvrAin the direct plate assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of
3935 µg/plate in the strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

Since in the first mutation assay, no toxicity and no precipitate on the plates was observed at the highest dose level tested in any of the five tester strains, an additional direct plate assay was performed. In the additional experiment the test item was tested at the dose level
5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

In the second mutation experiment, the test item was tested up to concentrations of
5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the
pre-incubation assay. The test item did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. 

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Bis-Aminopropyl Diglycol Dimaleate is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.