3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the chemical structure of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate, it can be concluded that, despite the positive in vitro micronucleus test, there is no concern for carcinogenicity in vivo. This was demonstrated by both absence of tumour formation in two carcinogenicity studies with maleic acid and maleic anhydride and absence of any reproductive toxicity related to genotoxic effects in a two-generation reproductive toxicity study with maleic anhydride. As a consequence, bis-aminopropyl diglycol dimaleate does not warrant classification for carcinogenicity according to GHS.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6/12/2017 to 15/2/2018

Reliability:

1 (reliable without restriction)

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

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

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:

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 / solvent:

The vehicle of the test item was Milli-Q water (Millipore Corp., Bedford, MA., USA).

Untreated negative controls:

yes

Negative solvent / vehicle 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 experimental 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.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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.

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 of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli(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 the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 November 2017 - 18 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Test item information
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear to transparent yellow liquid
Test item storage: At room temperature
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
CAS number: 11629579-82-3
Molecular formula: C18H32N2O11
Molecular weight: 452.46
pH ~ 3-4
Solubility in vehicle: Water: Highly
Stability in vehicle: Water: Stable

Target gene:

N/A

Species / strain / cell type:

lymphocytes: peripheral human

Details on mammalian cell type (if applicable):

Blood was collected from healthy adult, non-smoking volunteers (aged 18 to 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2017) are presented below:

Dose-range finding study: age 32, AGT = 14.8 h
First cytogenetic assay: age 35, AGT = 13.7 h
Cytogenetic assay 1A: age 26, AGT = 15.8 h
Cytogenetic assay 1B: age 32, AGT = 14.1 h (experiment with pH correction)
Cytogenetic assay 1C: age 26, AGT = 13.8 h (experiment with pH correction)
Second cytogenetic assay: age 23, AGT = 14.2 h

Cytokinesis block (if used):

Prior to the mitosis (during or after exposure of the test item) the chemical cytochalasin B was added to the cultures. Cytochalasin B arrests the formation of actin filaments. Consequently, the cell is not able to divide, but nuclear division still continues. In this way, cytochalasin B allows discrimination between cells that have undergone nuclear division (binucleated) and cells that have not (mononucleated).

Metabolic activation:

with and without

Metabolic activation system:

Metabolic Activation System
Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats (6), which were obtained from Charles River (Sulzfeld, Germany).

Preparation of S9-Mix
S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life Technologies).

The above solution was filter (0.22 m)-sterilized. To 0.5 mL S9-mix components 0.5 mL S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.

Metabolic activation was achieved by adding 0.2 mL S9-mix to 5.3 mL of a lymphocyte culture (containing 4.8 mL culture medium, 0.4 mL blood and 0.1 mL (9 mg/mL) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Test concentrations with justification for top dose:

FIRST CYTOGENETIC ASSAY
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
With and without S9-mix : 25, 250, 500, 600, 700, 800 and 900 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).

Both in the absence and presence of S9-mix, the test item did not induce a statistically significant increase in the number of mono- and binucleated cells with micronuclei . The results are therefore equivocal and the experiment was repeated in cytogenetic assay 1A:
With and without S9-mix : 250, 500, 600, 700, 800, 900 and 1000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).

To account for a possible effect of the increasing pH with increasing test item concentrations, an additional experiment was performed with the pH corrected to the solvent control level in experiment 1B:
With and without S9-mix : 100, 300, 600, 700, 800, 900 and 1000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).

In the presence of S9-mix, no appropriate dose level could be selected for scoring of micronuclei and therefore this experiment was repeated in cytogenetic assay 1C.
With S9-mix : 100, 500, 1000, 1250, 1500, 1750 and 2000 µg/mL culture medium (3 hours exposure time, 27 hours harvest time).


SECOND CYTOGENETIC ASSAY
To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 1, 10, 50, 75, 100, 125 and 150 µg/mL culture medium (24 hours exposure time, 24 hours harvest time).

Vehicle / solvent:

The vehicle for the test item was Milli-Q water
Solubility in vehicle: Highly

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Experimental dates: 02 January 2018 - 25 November 2018

PREPARATION OF TEST ITEM

A correction factor of 3.85 for the composition of the test item was applied in this study (based on the water content determined in Charles River Project 512944).
A solubility test was performed based on visual assessment. The test item formed a clear light yellow solution in Milli-Q water. Test item concentrations were used within 1 hour after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v). The pH and the osmolarity of the culture medium containing the highest tested concentration were recorded. The pH was also determined for additional concentrations (see study plan deviation). In the cytogenetic assay with pH correction (1B and 1C) the pH was measured for all concentrations and corrected with 1 M NaOH to the value of the solvent control. Any residual volumes were discarded.

CELL CULTURE

Blood samples
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Life Technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies), L-glutamine (2 mM) (Life Technologies), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) (Life Technologies) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel Europe Ltd., Dartford, United Kingdom) was added.

Environmental conditions
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 40 - 97%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.4 - 37.1°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

EXPERIMENTAL DESIGN

Dose-range Finding Test
In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose-range finding test. Bis-Aminopropyl Diglycol Dimaleate was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 mL blood of a healthy donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Bis-Aminopropyl Diglycol Dimaleate for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B (Sigma) was added to the cells simultaneously with the test item at the 24 hours exposure time. A vehicle control was included at each exposure time.
The highest tested concentration was 2000 µg/mL.
After 3 hours exposure to Bis-Aminopropyl Diglycol Dimaleate in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasine B (5 µg/mL) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately.
Cytotoxicity of Bis-Aminopropyl Diglycol Dimaleate in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.


First Cytogenetic Assay
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Bis-Aminopropyl Diglycol Dimaleate for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium with Cytochalasin B (5 µg/mL) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay. Additional experiments were performed since the results of the first cytogenetic assay were equivocal, and to correct for changes in the pH.

Second Cytogenetic Assay
To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Bis-Aminopropyl Diglycol Dimaleate with cytochalasin B (5 µg/mL) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay.

Preparation of Slides
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68 (Applichem, Darmstadt, Germany). After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethanol (Merck): acetic acid (Merck) fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol: acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

CONTROLS

Negative Control
The negative control used in the test system was the vehicle for the test item.

Positive Controls Without Metabolic Activation
Mitomycin C (MMC-C; CAS No. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct acting clastogen at a final concentration of 0.25 and 0.38 µg/mL for a 3 hour exposure period and 0.15 and 0.23 µg/mL for a 24 hour exposure period.
Colchicine (Colch; CAS No. 64-86-8, Acros Organics, Geel, Belgium) was used as a direct acting aneugen at a final concentration of 0.1 µg/mL for a 3 hour exposure period and
0.05 µg/mL for a 24 hour exposure period.

Positive Control With Metabolic Activation
Cyclophosphamide (CP; CAS No. 50-18-0, Baxter B.V., Utrecht, The Netherlands) was used as an indirect acting clastogen, requiring metabolic activation, at a final concentration of
15 and 17.5 µg/mL for a 3 hour exposure period.

Solvent for Positive Controls
Hanks’ Balanced Salt Solution (HBSS) (Life Technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

Rationale for test conditions:

The design of this study is based on the following study guideline:
OECD Guideline 487. In Vitro Mammalian Cell Micronucleus Test (adopted 29 July 2016).

Evaluation criteria:

Cytogenetic Assessment/Scoring of Micronuclei:

To prevent bias, all slides were randomly coded before examination of micronuclei and scored. An adhesive label with Charles River Den Bosch study identification number and code was stuck over the marked slide. At least 1000 (with a maximum deviation of 5%) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5%) mononucleated cells per culture were scored for micronuclei separately. Since the lowest concentration of MMC-C and CP resulted in a positive response the highest concentration was not examined for the presence of micronuclei in the first cytogenetic assay. In the second cytogenetic assay the highest concentration of MMC-C was scored. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000. However, when an expected statistical significant increase is observed, this has no effect on the study integrity.

The following criteria for scoring of binucleated cells were used (1 - 2, 6):
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996 (1):
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.


Cytotoxicity Assessment:
See 'Any other information' section

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

The Chi-square test showed that there are statistically significant differences between one or more of the test item groups and the vehicle control group. Therefore a Cochran Armitage trend test (p < 0.05) was performed to test whether there was a significant trend in the induction.

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

3 hours exposure time

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

3 hours exposure time

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

24 hours continuous exposure

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: peripheral human

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

24 hours continuous exposure

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING TEST

A concentration of 2000 µg/mL showed no precipitation in the culture medium and was used as the highest concentration of Bis-Aminopropyl Diglycol Dimaleate.
In the dose-range finding test blood cultures were treated with 125, 250, 500, 1000 and 2000 µg test item/mL culture medium and exposed for 3 and 24 hours in the absence of S9-mix and for 3 hours in the presence of S9-mix.The pH and osmolarity of a concentration of 2000 µg/mL were 5.9 and 315 mOsm/kg respectively (compared to 7.5 and 298 mOsm/kg in the solvent control).
Table 1 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the negative control item.


FIRST CYTOGENETIC ASASY

Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay:
With and without S9-mix : 25, 250, 500, 600, 700, 800 and 900 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

Table 2 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.
The following dose levels were selected for scoring of micronuclei:
Without S9-mix : 25, 500 and 800 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 25, 600 and 900 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

Although both in the absence and presence of S9-mix results above the 95% control limits of the distribution of the historical negative control database were observed at the intermediate and high concentration tested, both in the absence and presence of S9-mix, the test item did not induce a statistically significant increase in the number of mono- and binucleated cells with micronuclei (Appendix 1, Table 3). The results are therefore equivocal and the experiment was repeated in cytogenetic assay 1A:
With and without S9-mix : 250, 500, 600, 700, 800, 900 and 1000 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

The pH was measured of all concentrations tested at the end of the study. The pH of a concentration of 1000 µg/mL was 6.5 (compared to 7.5 in the solvent control). The concentrations of 900, 800, 700, 600, 500 and 250 µg/mL had a pH of 6.5, 6.6, 6.7, 6.8, 6.9 and 7.3 respectively.
Table 4 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.
The following dose levels were selected for scoring of micronuclei:
Without S9-mix : 600, 700 and 800 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 700, 800 and 900 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of mono- and binucleated cells with micronuclei at the highest concentration tested in the presence of S9-mix. In the absence of S9-mix, the test item induced a statistically significant increase in the number of mononucleated cells at the lowest and highest dose level tested and at all dose levels in the number of binucleated cells with micronuclei (Appendix 1, Table 5).
To account for a possible effect of the increasing pH with increasing test item concentrations, an additional experiment was performed with the pH corrected to the solvent control level in experiment 1B:
With and without S9-mix : 100, 300, 600, 700, 800, 900 and 1000 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).

Table 6 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.
In the presence of S9-mix, no appropriate dose level could be selected for scoring of micronuclei and therefore this experiment was repeated in cytogenetic assay 1C.
With S9-mix : 100, 500, 1000, 1250, 1500, 1750 and 2000 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
The pH was measured of all concentrations tested and corrected with 1M NaOH to values between 7.2 and 7.3 (see table below). The pH of the solvent control was 7.3.

Table 7 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.
The following dose levels were selected for scoring of micronuclei:
Without S9-mix : 100, 600 and 800 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
With S9-mix : 100, 1000 and 1500 µg/mL culture medium
(3 hours exposure time, 27 hours harvest time).
The pH was measured of all concentrations tested and corrected with 1M NaOH to values between 7.7 and 7.8 (see table next page). The pH of the solvent control was 7.8.

Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of binucleated cells with micronuclei at all concentrations tested in the absence of
S9-mix (Appendix 1, Table 8). These increases were all within the historical control data range. In the presence of S9-mix, the test item induced a statistically significant increase in the number of mononucleated cells with micronuclei at the highest concentration tested. Additionally, the test item induced a statistically significant increase in the number of binucleated cells with micronuclei at all concentrations tested. The increase in the number of binucleated cells is above the historical control data range at the highest concentration tested.

SECOND CYTOGENETIC ASASY

To obtain more information about the possible clastogenicity and aneugenicity of the test item, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 1, 10, 50, 75, 100, 125 and 150 µg/mL culture medium
(24 hours exposure time, 24 hours harvest time).
Table 9 shows the cytokinesis-block proliferation index of cultures treated with various test item concentrations or with the positive or negative control items.

The following dose levels were selected for the scoring of micronuclei:
Without S9-mix : 10, 100, 125 and 150 µg/mL culture medium
(24 hours exposure time, 24 hours harvest time).
Bis-Aminopropyl Diglycol Dimaleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei (Appendix 1; Table 10).

DISCUSSION

The ability of Bis-Aminopropyl Diglycol Dimaleate to induce micronuclei in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analyzed was selected based on toxicity, cytokinesis-block proliferation index of 55 ± 5%. 

The cytokinesis-block proliferation indices of cultures treated with various Bis-Aminopropyl Diglycol Dimaleate concentrations or with the negative control items are presented in
Table1,Table2,Table4,Table6,Table7andTable9(Appendix 1). The scores for the number of mono- and binucleated cells with micronuclei are presented inTable3,Table5,Table8andTable10(Appendix 1). Duplicate cultures are indicated by A and B. The individual data are described inTable11-Table15Table15(Appendix 2). Appendix 3presents the statistical evaluations of the test results.

The number of mononucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database. (Table3,Table5,Table8andTable10;Appendix 4). 

The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system
(S9-mix) functioned properly. 

In the first cytogenetic assay, both in the absence and presence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate did not induce a statistically significant increase in the number of mono- and binucleated cells with micronuclei. However, results above the 95% control limits of the distribution of the historical negative control database were observed at the intermediate and high concentration tested, both in the absence and presence of S9-mix. 

Due to the equivocal results a repeat experiment was performed. In this repeat experiment, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of mono- and binucleated cells with micronuclei at the highest concentration tested in the presence of S9-mix and at 2 or 3 concentrations tested in the absence of S9-mix. A statistical significant dose related trend was observed both in the absence and presence of S9-mix and both in the number of mono and binucleated cells with micronuclei. Although a statistical significant dose related trend was observed the results observed in the presence of S9-mix and the results obtained for the number of mononucleated cells with micronuclei in the absence of S9-mix (mean number of micronuclei) are within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant.

The statistically significant increase in the number of binucleated cells with micronuclei in the absence of S9-mix was above the 95% control limits of the distribution of the historical negative control database at the lowest and highest concentration tested. Moreover a statistical significant dose related trend was observed and therefore this increase was considered biologically relevant. However, care should be taken with the interpretation of the results since the pH of the test item was low in comparison with the solvent control. The low pH may lead to artifactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes. Therefore, additional experiments with pH correction were performed to account for this possible effect.

In the additional cytogenetic assays with correction of the pH, in the absence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of binucleated cells with micronuclei. Although a statistical significant dose related trend was observed the results (mean number of micronuclei) are well within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant. In the presence of S9-mix, at the 3 hours exposure time, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of mono- and binucleated cells with micronuclei. Although a statistical significant dose related trend was observed for the number of mononucleated cells with micronuclei the results (mean number of micronuclei) are within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant. For the number of binucleated cells with micronuclei, the test item induced a statistically significant and dose dependent increase, which was considered to be biologically relevant since the results were outside the 95% control limits of the distribution of the historical negative control database.

In the second cytogenetic assay with a 24 hours continuous exposure time in the absence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

Conclusions:

In conclusion, this test is valid and Bis-Aminopropyl Diglycol Dimaleate induces the formation of micronuclei in binucleated cells in human lymphocytes in the absence of S9 metabolic activation, under the experimental conditions described in this report. In contrast, in the presence of S9-mix no such genotoxic effect was found. This positive response was observed without a correction for the pH of the culture medium.

After correction for the pH, Bis-Aminopropyl Diglycol Dimaleate induces the formation of micronuclei in binucleated cells in human lymphocytes in the presence of S9 mix. In contrast, in the absence of S9-mix no such genotoxic effect was found. Since Bis-Aminopropyl Diglycol Dimaleate induces the micronuclei frequency in binucleated cells, it may be considered a clastogenic compound.

Executive summary:

The objective of this study was to evaluate Bis-Aminopropyl Diglycol Dimaleate for its ability to induce micronuclei in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix). The possible clastogenicity and aneugenicity of Bis-Aminopropyl Diglycol Dimaleate was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guideline.

The sample of Bis-Aminopropyl Diglycol Dimaleate was a clear to transparent yellow liquid, which contains 74% water.A correction factor of 3.85 was used to correct for the composition. The vehicle of the test item was Milli-Q water.

In the first cytogenetic assay, Bis-Aminopropyl Diglycol Dimaleate was tested for a 3 hours exposure time with a 27 hours harvest time up to 800 µg/mL in the absence and up to 900 µg/mL in the presence of S9-fraction. Appropriate toxicity was reached at these dose levels. Since the results of the first cytogenetic assay were equivocal an additional experiment was performed. In cytogenetic assay 1A the test item was again tested up to800 and 900 µg/mL in the absence and presence of S9-mix, respectively.

In the second cytogenetic assay, Bis-Aminopropyl Diglycol Dimaleate was tested up to 150 µg/mL for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The number of mononucleated cells with micronuclei found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition, the number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system(S9-mix) functioned properly.

In the first cytogenetic assay, both in the absence and presence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate did not induce a statistically significant increase in the number of mono- and binucleated cells with micronuclei. However, results above the 95% control limits of the distribution of the historical negative control database were observed at the intermediate and high concentration tested, both in the absence and presence of S9-mix. 

Due to the equivocal results a repeat experiment was performed. In this repeat experiment, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of mono- and binucleated cells with micronuclei at the highest concentration tested in the presence of S9-mix and at 2 or 3 concentrations tested in the absence of S9-mix. A statistical significant dose related trend was observed both in the absence and presence of
S9-mix and both in the number of mono and binucleated cells with micronuclei. Although a statistical significant dose related trend was observed the results observed in the presence of S9-mix and the results obtained for the number of mononucleated cells with micronuclei in the absence of S9-mix (mean number of micronuclei) are within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant.

The statistically significant increase in the number of binucleated cells with micronuclei in the absence of S9-mix was above the 95% control limits of the distribution of the historical negative control database at the lowest and highest concentration tested. Moreover a statistical significant dose related trend was observed and therefore this increase was considered biologically relevant. However, care should be taken with the interpretation of the results since the pH of the test item was low in comparison with the solvent control. The low pH may lead to artifactual positive results, i.e. chromosome damage not caused by direct interaction between the test item and chromosomes. Therefore, additional experiments with pH correction were performed to account for this possible effect.

In the additional cytogenetic assays with correction of the pH, in the absence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of binucleated cells with micronuclei. Although a statistical significant dose related trend was observed the results (mean number of micronuclei) are well within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant. In the presence of S9-mix, at the 3 hours exposure time, Bis-Aminopropyl Diglycol Dimaleate induced a statistically significant increase in the number of mono- and binucleated cells with micronuclei. Although a statistical significant dose related trend was observed for the number of mononucleated cells with micronuclei the results (mean number of micronuclei) are within the 95% control limits of the distribution of the historical negative control database and therefore these increases were considered not biologically relevant. For the number of binucleated cells with micronuclei, the test item induced a statistically significant and dose dependent increase, which was considered to be biologically relevant since the results were outside the 95% control limits of the distribution of the historical negative control database.

In the second cytogenetic assay with a 24 hours continuous exposure time in the absence of S9-mix, Bis-Aminopropyl Diglycol Dimaleate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

In conclusion, this test is valid and Bis-Aminopropyl Diglycol Dimaleate induces the formation of micronuclei in binucleated cells in human lymphocytes in the absence of S9 metabolic activation, under the experimental conditions described in this report. In contrast, in the presence of S9-mix no such genotoxic effect was found. This positive response was observed without a correction for the pH of the culture medium. 

After correction for the pH, Bis-Aminopropyl Diglycol Dimaleate induces the formation of micronuclei in binucleated cells in human lymphocytes in the presence of S9 mix. In contrast, in the absence of S9-mix no such genotoxic effect was found. Since Bis-Aminopropyl Diglycol Dimaleate induces the micronuclei frequency in binucleated cells, it may be considered a clastogenic compound. 

Reference 3

Endpoint:

genetic toxicity in vitro, other

Remarks:

Weight of Evidence - genotoxicity of bis-aminopropyl diglycol dimaleate (CAS No. 1629579-82-3)

Type of information:

other: Weight of Evidence - genotoxicity of bis-aminopropyl diglycol dimaleate (CAS No. 1629579-82-3)

Adequacy of study:

weight of evidence

Study period:

08 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 - Weight of Evidence report

Qualifier:

no guideline required

Principles of method if other than guideline:

A Weight of Evidence approach has been used to address this endpoint, information based on the chemical structure of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate were assesssed.

GLP compliance:

no

Remarks:

Not required

Type of assay:

other: Weight of Evidence report - Review of a negative Ames test and a positive in vitro micronucleus assay identifying bis-aminopropyl diglycol dimaleate as a potential clastogenic substance.

Specific details on test material used for the study:

Test Item Name: Bis-Aminopropyl Diglycol Dimaleate
CAS Number: 1629579-82-3

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

DISCUSSION

Available toxicity data for bis-aminopropyl diglycol dimaleate consists of a negative Ames test and a positive in vitro micronucleus assay identifying bis-aminopropyl diglycol dimaleate as a potential clastogenic substance. Based on the structure and the reactivity of the components present in bis-aminopropyl diglycol dimaleate, the maleate groups are expected to be responsible for the effects observed in the in vitro micronucleus assay. Maleic acid and maleic anhydride were negative in the Ames test similar to bis-aminopropyl diglycol dimaleate. In vitro micronucleus data are not available for maleic acid or maleic anhydride, but the in vitro chromosome aberration test performed with maleic anhydride was positive, identifying a similar concern for clastogenic potential for maleic acid/maleic anhydride based on available in vitro genotoxicity data. However, the in vivo carcinogenicity tests for maleic acid and maleic anhydride were negative. In addition, the two-generation reproductive toxicity study with maleic anhydride did not reveal any concern for reduced fertility or genetic effects in offspring. Based on the chemical structure, the reactivity of the individual components of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate, it can be concluded that, despite the positive in vitro micronucleus test, there is no concern for carcinogenicity in vivo.

Conclusions:

Based on the chemical structure of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate, it can be concluded that, despite the positive in vitro micronucleus test, there is no concern for carcinogenicity in vivo. This was demonstrated by both absence of tumour formation in two carcinogenicity studies with maleic acid and maleic anhydride and absence of any reproductive toxicity related to genotoxic effects in a two-generation reproductive toxicity study with maleic anhydride. As a consequence, bis-aminopropyl diglycol dimaleate does not warrant classification for carcinogenicity according to GHS.

Executive summary:

The objective of this evaluation was to reach an overall conclusion on genotoxic potential of products containing bis-aminopropyl diglycol dimaleate. Studies provided by the sponsor addressing the genotoxicity endpoint performed with bis-aminopropyl diglycol dimaleate were evaluated.

Available data for bis-aminopropyl diglycol dimaleate consists of a negative Ames test and a positive in vitro micronucleus assay identifying bis-aminopropyl diglycol dimaleate as a potential clastogenic substance. Based on the chemical structure and the reactivity of the individual components of bis-amino diglycol dimaleate, the maleate groups are likely responsible for observed effects. Relevant data included in the OECD SIDS document for maleic acid and maleic anhydride was therefore evaluated for the observed effects in the genotoxicity studies and integrated with the available data for bis-aminopropyl diglycol dimaleate in a weight of evidence approach.

A similar concern for clastogenic potential was identified for maleic anhydride which was positive in the in vitro chromosome aberration test. However, the in vivo carcinogenicity tests for maleic acid and maleic anhydride were negative. In addition, the two-generation reproductive toxicity study with maleic anhydride did not reveal any concern for reduced fertility or genetic effects in offspring.

Based on the chemical structure of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate, it can be concluded that, despite the positive in vitro micronucleus test, there is no concern for carcinogenicity in vivo as demonstrated by both absence of tumour formation in two carcinogenicity studies with maleic acid and maleic anhydride and absence of any reproductive effect related to genotoxic effect in a two-generation reproductive toxicity study with maleic acid. As a consequence, bis-aminopropyl diglycol dimaleate does not warrant classification for carcinogenicity according to GHS.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

other justification

Justification for data waiving:

other:

Justification for type of information:

Weight of Evidence CRL 2020: Based on the chemical structure of bis-aminopropyl diglycol dimaleate and similarity in effects observed in vitro between maleic acid/maleic anhydride and bis-aminopropyl diglycol dimaleate, it can be concluded that, despite the positive in vitro micronucleus test, there is no concern for carcinogenicity in vivo. This was demonstrated by both absence of tumour formation in two carcinogenicity studies with maleic acid and maleic anhydride and absence of any reproductive toxicity related to genotoxic effects in a two-generation reproductive toxicity study with maleic anhydride. As a consequence, bis-aminopropyl diglycol dimaleate does not warrant classification for carcinogenicity according to GHS.

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

Bis-aminopropyl diglycol dimaleate does not warrant classification for carcinogenicity according to GHS.