Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Test, Groot (2018)

Under the conditions of the study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 March 2018 to 19 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames]
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD, EC).
- The characteristics of the different Salmonella typhimurium strains were as follows:
TA1537 hisC3076 Frameshift,
TA98 hisD3052/R-factor Frameshift,
TA1535 hisG46 Base-pair substitutions,
TA100 hisG46/R-factor Base-pair substitutions.
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 and
uvrB: loss of the excision repair system (deletion of the ultraviolet-repair B gene).
- The Salmonella typhimurium strains are checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.
- Stock cultures were stored in liquid nitrogen (-196 °C).
- 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 (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH, Germany [Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK].
- 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 permeabilisation of the strain using Tris-EDTA treatment (Ref.1). The strain is checked to confirm the tryptophan-requirement, UV-sensitivity and the number of spontaneous revertants at least every year.
- Stock cultures were stored in liquid nitrogen (-196 °C).
- 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 (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

First Experiment: Based on the results of the dose-range finding test, the following dose-range was selected for the first mutation experiment: 52, 164, 512, 1600 and 5000 µg/plate
Second Experiment: Based on the results of the first mutation assay, the test material was tested up to the dose level of 5000 μg/plate (492, 878, 1568, 2800 and 5000 µg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material formed a yellow to orange homogenous suspension in ethanol at 50 mg/mL. At concentrations of 28 mg/mL and below, the test material was soluble in ethanol. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test material concentrations were used within 2 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 for TA1537 -S9 mix at 2.5 µg/plate and 2-aminoanthracene for all strains +S9 mix (1 - 15 µg/plate)

Details on test system and experimental conditions:

DOSE-RANGE FINDING TEST
- 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 5 % (v/v) S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of the test material used in the subsequent mutation assays was 5000 μg/plate.

METHOD OF APPLICATION: in agar

DURATION
- Exposure duration: 48 ± 4 hours

NUMBER OF REPLICATIONS: 3

METHOD
- At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain. The above mentioned dose-range finding study with the two tester strains TA100 and WP2uvrA, is reported as a part of the first mutation experiment. In the second part of this experiment, the test material was tested both in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. In a follow-up experiment with additional parameters, the test material was tested both in the absence and presence of 10 % (v/v) S9-mix in all tester strains.
- The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.
- Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

COLONY COUNTING
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

INTERPRETATION
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

- A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535 & TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

observed in the tester strains TA1535 (presence of S9-mix) and TA98 (absence of S9-mix) at the highest dose level tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

DOSE-RANGE FINDING TEST/ FIRST MUTATION EXPERIMENT
- The test material was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 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 experiment with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.
- Precipitate: Precipitation of the test material on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined. No reduction of the bacterial background lawn was observed. Cytotoxicity, as evidenced by a decrease in the number of revertants was only observed in the tester strains TA1535 (presence of S9-mix) and TA98 (absence of S9-mix) at the highest dose level tested.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test material under all conditions tested.

SECOND MUTATION EXPERIMENT
- To obtain more information about the possible mutagenicity of the test material, a second mutation experiment was performed in the absence and presence of 10 % (v/v) S9-mix. Based on the results of the first mutation assay, the test material was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA.
- Precipitate: Precipitation of the test material on the plates was observed at the start and at the end of the incubation period at concentrations of 1568 μg/plate and above.
- Toxicity: In the second mutation assay, there was no reduction of 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 second mutation assay, no increase in the number of revertants was observed upon treatment with the test material under all conditions tested.
- In tester strain TA1537 in the presence of S9-mix, the test material induced up to 4.7-fold increase in the number of revertant colonies compared to the solvent control. However no dose relation was observed and the increases were within the historical control data range. Therefore, these increases were considered to be not biologically relevant.

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 experiments.
The negative and 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.

Table 1: Summary of Experiment 1

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 1.7 5.4 17 52 164 512 1600 5000	137 130 110 96 116 104 118 106 NP 103 n SP	7 - - - 13 8 8 9 NP 6 n SP	19 19 21 13 16 16 20 17 NP 19 n SP	12 - - - 12 15 13 15 NP 5 n SP	4 - - - 4 3 3 2 NP 5 n SP
+	Solvent 1.7 5.4 17 52 164 512 1600 5000	131 110 101 109 102 95 97 91 NP 69 n SP	12 - - - 10 11 16 16 NP 4 n SP	21 29 26 23 27 22 21 24 NP 16 n SP	22 - - - 21 24 19 15 NP 12 n SP	3 - - - 6 8 8 5 NP 7 n SP
Positive Controls
-	Name	MMS	SA	4-NQO	2-NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	967	1160	1584	1349	970
+	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration (µg/plate)	1	2.5	15	1	2.5
	Mean no. colonies/plate	1508	399	441	987	224

4-NQO = 4-Nitroquinoline-1-oxide

2-AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

MMS = methylmethanesulfonate

ICR-191

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

Results with metabolic activation: Plate incorporation assay (5 % S9)

Table 2: Summary of Experiment 2

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		Base-pair Substitution Type			Frameshift Type
		TA100	TA1535	WP2uvrA	TA98	TA1537
-	Solvent 492 878 1568 2800 5000	108 102 101 NP 109 SP 87 SP 106 n MP	12 11 18 NP 8 SP 9 SP 12 n MP	30 37 31 NP 30 SP 22 SP 29 n MP	17 19 15 NP 19 SP 15 SP 18 n MP	3 5 7 NP 6 SP 7 SP 8 n MP
+	Solvent 492 878 1568 2800 5000	60 72 77 NP 70 SP 75 SP 81 n MP	14 15 12 NP 9 SP 14 SP 14 n MP	49 40 39 NP 31 SP 34 SP 37 n MP	29 36 37 NP 42 SP 32 SP 25 n MP	3 14 11 NP 7 SP 10 SP 8 n MP
Positive Controls
-	Name	MMS	SA	4-NQO	2-NF	ICR-191
	Concentration (µg/plate)	650	5	10	10	2.5
	Mean no. colonies/plate	1068	1045	1153	1213	926
+	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentration (µg/plate)	2	2.5	15	1	5
	Mean no. colonies/plate	1041	283	389	517	288

4-NQO = 4-Nitroquinoline-1-oxide

2-AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

MMS = methylmethanesulfonate

ICR-191

MP = Moderate Precipitate

NP = No precipitate

SP = Slight Precipitate

n = Normal bacterial background lawn

Results with metabolic activation: Plate incorporation assay (10 % S9)

Conclusions:

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions.

The objective of this study was to determine the potential of the test material 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 WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at the dose level of 5000 μg/plate. 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.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. SME-50 precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in the tester strains TA1535 (presence of S9-mix) and TA98 (absence of S9-mix) at the highest dose level tested.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 1568 μg/plate and above. 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.

The test material 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and 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.

Under the conditions of this study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test, Groot (2018)

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B.13/14, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the potential of the test material 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 WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

In the dose-range finding test, the test material was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test material precipitated on the plates at the dose level of 5000 μg/plate. 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.

Based on the results of the dose-range finding test, the test material was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5 % (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. SME-50 precipitated on the plates at the top dose of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants was observed in the tester strains TA1535 (presence of S9-mix) and TA98 (absence of S9-mix) at the highest dose level tested.

In a follow-up experiment of the assay with additional parameters, the test material was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test material precipitated on the plates at dose levels of 1568 μg/plate and above. 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.

The test material 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 the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

The negative and 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.

Under the conditions of the study, the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.