Dipropylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- AMES (OECD 471): negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 December 2021-20 December 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 B13/14

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

June 26, 2020

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital/beta-naphthoflavone induced rat liver S9
- method of preparation of S9 mix: The S9 was prepared and stored according to the currently valid version of the SOP for rat liver S9 preparation.
- concentration or volume of S9 mix and S9 in the final culture medium: The protein concentration of the S9 preparation was 29.6 mg/mL (Lot. No.: 080721D). An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution, to result in a final concentration of approx. 10% (v/v) in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed six concentrations were tested in experiment II 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Since the acceptance criterion of at least five analysable concentrations was not fulfilled in experiment II in strains TA 98 and TA 100 without S9 mix because of strong toxicity, this part was repeated as a pre-incubation assay with the following concentrations (reported as experiment IIa):
10; 33; 100; 333; 1000; and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria (Maron et al.; 1981).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD and 2-Aminoanthracene

Remarks:

In parallel to each test a sterile control of the test item was performed. 100 µL of the stock solution, 500 µL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration : triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I since the acceptance criteria are met.

In the pre-experiment the concentration range of the test item was 3 – 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed six concentrations were tested in experiment II 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades.
The following concentrations were tested in experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since the acceptance criterion of at least five analysable concentrations was not fulfilled in experiment II in strains TA 98 and TA 100 without S9 mix, this part was repeated as a pre-incubation assay with the following concentrations (reported as experiment IIa): 10; 33; 100; 333; 1000; and 2500 µg/plate

Rationale for test conditions:

To establish a dose response effect at least six dose levels with adequately spaced concentrations were tested. The maximum dose level was 5000 µg/plate in experiment I and II. Based on the toxicity, observed in experiment II a lower top dose of 2500 µg/plate was chosen in experiment IIa.

Evaluation criteria:

Acceptability of the Assay:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent control;
• the spontaneous reversion rates in the negative and solvent control are in the range of our historical data;
• the positive control substances should produce an increase above the threshold of twofold (strains TA 98, TA 100, and WP2 uvrA) or threefold (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation of Results:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

evident as reduction in the number of revertants

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:

cytotoxicity

Remarks:

evident as reduction in the number of revertants

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:

cytotoxicity

Remarks:

evident as reduction in the number of revertants

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:

cytotoxicity

Remarks:

evident as reduction in the number of revertants

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:

cytotoxicity

Remarks:

evident as reduction in the number of revertants

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation S9 mix, experiment IIa was performed without S9 mix, only. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since the acceptance criterion of at least five analysable concentrations was not fulfilled in experiment II in strains TA 98 and TA 100 without S9 mix , this part was repeated (experiment IIa) at the following concentrations:
10; 33; 100; 333; 1000; and 2500 µg/plate
No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate.
The plates incubated with the test item showed reduced background growth in experiment I in strains TA 1537 and TA 98 at 5000 µg/plate with and without S9 mix.
Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in experiment 1 in strain TA 1537 in the in the absence of S9 mix at 5000 µg/plate, in experiment II in all strains used from 2500 to 5000 µg/plate in the absence of S9 mix and at 5000 µg/plate in the presence of S9 mix and in experiment IIa in strains TA 98 and TA 100 in the absence of S9 mix at 2500 µg/plate.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Dipropylamine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.



TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The pH was monitored for all concentrations, which were obtained by serial dilution. A slightly increased pH value (> pH 10) was observed at concentrations of 2500 µg/plate and above in experiment I and II and in experiment IIa at a concentration of 1000 µg/plate and above.
- Precipitation and time of the determination: No precipitation of the test item occurred in the overlay agar in the test tubes. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I at 5000 µg/plate. Due to precipitation of the test item the colonies were partly counted manually.


Ames test:
- Signs of toxicity
- Individual plate counts
- Mean number of revertant colonies per plate and standard deviation

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II		Experiment IIa
	without S9 mix	with S9 mix	without S9 mix	with S9 mix	without S9 mix
TA 1535	/	/	2500 – 5000	5000	n.p.
TA 1537	5000	/	2500 - 5000	5000	n.p.
TA 98	/	/	2500 – 5000	5000	2500
TA 100	/	/	2500 – 5000	5000	2500
WP2 uvrA	/	/	2500 – 5000	5000	n.p.

/ = no toxic effects (induction factor ≥ 0.5)

n.p. = not performed

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Dipropylamine at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.

Conclusions:

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Dipropylamine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well documented publication which meets basic scientific principles.

Principles of method if other than guideline:

according to Haworth, S. et al.: Environ. Mutagen. 5, Suppl. 1, 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

induced male Sprague Dawley rat liver S9, induced male Syrian hamster liver S9

Test concentrations with justification for top dose:

33, 100, 333, 1000 und 3333 µg/Platte

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: For strains tested in the absence of S9: TA98, 2-nitrofluorene, TA100 and TA1535, sodium azide, TA1537, 9-aminoacridine. For strains tested with S9: All strains, 2-aminoanthracene.

Details on test system and experimental conditions:

IUCLID4 Type: Ames test
METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 100 at 3333 µg

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Strain 1535

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	18	1.2	8	1.5	17	3	9	0.6	20	1.5	8	0.7
33	20	3.2	7	1.5	19	2	8	1.7	20	0.3	7	2.6
100	19	1.7	5	0.7	19	1	8	0.7	17	1.9	6	2.6
333	16	1.2	7	0.6	20	1.2	6	0.9	25	1.2	5	1.2
1000	17	2.3	6	1.7	16	2.3	7	1.2	20	2.4	7	1.7
3333	13	2.7	3	1.2	23	3.3	9	1.2	27	0.6	9	0.7
Positive Control	1224	75.1	522	28.9	298	12.2	213	11.7	260	9.3	170	20.8

Strain: TA100

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	160	3.5	133	9.8	164	12.1	178	15.9	195	8.8	162	9.8
33	150	3.5	133	2.2	176	12.9	157	5.8	186	3.6	156	4.2
100	155	6.9	120	9.4	176	13.4	160	11.7	196	4.6	175	13.1
333	144	2.1	131	4.7	187	2.6	174	6.7	200	5.1	124	8.1
1000	151	9.2	125	4.3	174	20.2	125	5.6	157	20.5	108	6.7
3333	t		123	2.6	t		80	7.3	t		92	1.2
Positive Control	1707	21.1	1393	35.5	2583	115.7	1536	96.6	1946	104.3	1563	85.9

 

  

Strain TA 98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	19	4.6	11	0.6	26	2.7	16	3.5	28	4.3	18	0.9
33	16	3.1	7	1.8	14	1.9	12	0.7	18	3.4	13	1.3
100	20	1.3	10	0.9	12	1.9	11	0.7	18	1.5	13	0.9
333	12	0.7	12	2.5	19	0.3	9	3	19	1.5	12	2.2
1000	18	1.9	6	1	16	1.5	11	1	17	3.5	10	1
3333	18	3.1	11	0.6	19	2.3	8	0	16	2.3	10	0.9
Positive Control	259	10	156	2.5	1322	66.6	1297	59.5	984	19.7	916	203.8

Strain: TA1537

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	9	0.9	9	1.2	12	2.7	8	2.2	13	1	11	0.6
33	6	1.5	5	1.3	17	0.9	10	2.3	10	1.8	7	2.2
100	11	2.1	4	0.9	18	2.3	8	0.9	11	1.2	5	0.9
333	7	1.2	5	1.7	16	2.7	6	2	10	0.9	7	0.7
1000	6	1.2	7	2.3	12	2.1	7	1.2	14	3.3	5	0.6
3333	7	1	7	2.3	11	1.9	4	2	9	2.9	6	0.7
Positive Control	888	239.7	141	22.5	184	42.5	165	2.7	500	11.3	218	5

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Under the conditions tested the test substance was non mutagenic in the bacterial AMES-Test. The positive control gave the expected values.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a GLP-compliant reverse mutation assay (OECD 471). The test substance was tested at doses of 3.3 µg - 5000 µg/plate in different strains (TA 1535, TA 100,TA 1537, TA 98 and E.coli WP2 uvrA) with and without metabolic activation (liver S9 mix). The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation (2022, 1).

 

Dipropylamine was evaluated for mutagenicity in the Salmonella/microsome preincubation assay using a standard protocol approved by the National Toxicology Program. Dipropylamine doses of 0, 33, 100, 333, 1000 and 3333 µg/plate were tested in four Salmonella typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537) in the presence and absence of Aroclor-induced rat or hamster liver S9. These tests were negative and the highest ineffective dose level tested in all four Salmonella tester strains under all treatment conditions was 3333 µg/plate. (Zeiger et al. 1988; 2).

Short description of key information:
In vitro studies:
Ames-Test: negative (OECD 471 and NTP standard protocol)

In vivo studies:
No valid data available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

A valid Ames test, with or without metabolic activation through S9-Mix, failed to provide any evidence for a mutagenic effect of Dipropylamine. The test substance is not warranted for classification.