N-Phenyl-diethanolamine, reaction products with formaldehyde

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 March 2020 to 16 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The test was conducted in accordance with the relevant OECD test guideline and in accordance with GLP. All validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Storage conditions: At room temperature protected from light
Physical description: Light orange liquid

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain 2

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
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
- method of preparation of S9 mix
S9-mix was prepared immediately before use and kept refrigerated. 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.

Test concentrations with justification for top dose:

In the first mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the strains TA1535, TA1537 and TA98. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.
In the second mutation experiment, the test item was tested up to concentrations of
1600 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 μg/plate in the tester strain WP2uvrA in the pre-incubation assay. The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (Merck, Darmstadt, Germany)
Saline = physiological saline (Eurovet Animal Health, Bladel, The Netherlands), used for TA1535 without metabolic activation

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 4-nitroquinoline N-oxide; 2-aminoanthracene

Details on test system and experimental conditions:

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

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1E9 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 30 ± 2 minutes (pre-incubation assays only)
- Exposure duration/duration of treatment: 48 ± 4 h
- Harvest time after the end of treatment (sampling/recovery times): Not specified


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- background growth inhibition; decreased in number of revertants and/or presence of microcolonies
- Any supplementary information relevant to cytotoxicity:

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- dose-related increase in the number of revertant colonies compared to the solvent control

Evaluation criteria:

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

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three times the concurrent control.
b) In case a repeat 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Dose-Range Finding Test:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100	WP2uvrA

Without S9-mix

Positive control	773	±	176		1418	±	112
Solvent control	120	±	10		13	±	1
1.7	112	±	14		17	±	5
5.4	118	±	25		23	±	8
17	123	±	14		20	±	7
52	123	±	23		19	±	5
164	115	±	25		26	±	4
512	113	±	8	^n	20	±	3
1600				^e MC	18	±	4	^n
5000	0	±	0	^a NP				^e NP  MC

With S9-mix

Positive control	1104	±	304		297	±	84
Solvent control	88	±	8		22	±	7
1.7	121	±	30		22	±	6
5.4	119	±	18		20	±	2
17	110	±	30		20	±	9
52	117	±	6		20	±	2
164	93	±	26		26	±	4
512	118	±	34	^n	27	±	6
1600				^e MC	39	±	6	^n
5000	0	±	0	^a NP	25	±	5	^s NP

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Table 2          
Experiment 1:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay

Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
	TA1535	TA1537	TA98

Without S9-mix

Positive control	1014	±	53		1120	±	9		1292	±	136
Solvent control	9	±	3		6	±	5		10	±	1
5.4	11	±	8		2	±	2		13	±	5
17	8	±	2		6	±	4		9	±	4
52	9	±	5		4	±	4		8	±	2
164	10	±	2		9	±	7		13	±	3
512	11	±	9	^n	6	±	4	^n	23	±	5	^n
1600	5	±	2	^m NP	4	±	1	^m NP				^e NP  MC

With S9-mix

Positive control	354	±	24		311	±	23		1656	±	337
Solvent control	15	±	11		5	±	3		16	±	3
5.4	11	±	4		6	±	6		21	±	3
17	15	±	8		7	±	3		16	±	2
52	14	±	4		6	±	4		17	±	5
164	9	±	4		7	±	5		18	±	3
512	16	±	6	^n	7	±	0	^n	26	±	7	^n
1600	7	±	4	^m NP				^e NP  MC	27	±	13	^s NP

 

MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

Table 3          
Experiment 2:  Mutagenic Response of the test item in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay

Pre-incubation Assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535	TA1537	TA98	TA100	WP2uvrA

Without S9-mix

Positive control	907	±	31		136	±	13		1879	±	59		558	±	68		1818	±	34
Solvent control	4	±	0		7	±	1		12	±	2		92	±	9		15	±	4
5.4	8	±	0		7	±	2		5	±	4		83	±	9			-
17	7	±	6		7	±	4		6	±	3		103	±	13		16	±	7
52	8	±	5		10	±	3		20	±	6		192	±	11		15	±	4
164	12	±	5	^n	5	±	0	^n	40	±	6	^n	274	±	39	^n	23	±	4
512	15	±	4	^s	5	±	2	^s	16	±	3	^s	83	±	30	^s	46	±	8	^n
1600				^a NP				^e NP  MC				^a NP				^a NP	9	±	6	^s
5000		-				-				-				-						^a NP

With S9-mix

Positive control	154	±	14		61	±	15		403	±	20		1088	±	28		536	±	16
Solvent control	7	±	2		4	±	3		13	±	6		56	±	4		18	±	6
5.4	11	±	6		6	±	2		19	±	4		83	±	20			-
17	7	±	4		9	±	2		18	±	10		78	±	8		18	±	2
52	8	±	4		7	±	3		30	±	10		100	±	8		23	±	8
164	13	±	7	^n	4	±	3	^n	58	±	4		184	±	60	^n	31	±	4
512	13	±	2	^s	3	±	1	^s	46	±	15	^n	96	±	6	^s	52	±	4	^n
1600				^e NP  MC				^e NP  MC				^e NP  MC				^a NP	7	±	4	^s
5000		-				-				-				-						^a NP

 

 

MC	Microcolonies
NP	No precipitate
a	Bacterial background lawn absent
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced
-	Not tested

 

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results of this study it is concluded that the test item is 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 the test item and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonellatyphimurium (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). 

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP₂uvrA in the direct plate assay.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in both tester strains in the absence and presence of S9-mix. 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
1600 µg/plate in the strains TA1535, TA1537 and TA98.  The test item did not precipitate on the plates at this dose level.  Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

In the second mutation experiment, the test item was tested up to concentrations of
1600 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and up to 5000 µg/plate in the tester strain WP₂uvrA in the pre-incubation assay.  The test item did not precipitate on the plates. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix. 

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.

In the second experiment (pre-incubation method), the test item induced dose-related increases in the number of revertant colonies compared to the solvent control in tester strains TA98, TA100 and WP₂uvrA

In tester strain TA98, the increases observed where up to 3.3- fold and 4.5- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain TA100, the increases observed where up to 3.0- fold and 3.3- fold the concurrent control, in the absence and presence of S9-mix, respectively.

In tester strain WP₂uvrA, the increases observed where up to 3.1- fold and 2.9- fold the concurrent control, in the absence and presence of S9-mix, respectively.

Although the increases in tester strains TA98 and WP₂uvrA were within the laboratory historical control data range, the responses were more than two- and three-fold the concurrent controls in strains WP₂uvrA and TA98, respectively. In addition, the increases in tester strain TA100 were outside the historical control data range and more than two-fold the concurrent controls. Based on these observations, the increases were considered biologically relevant. 

In conclusion, based on the results of this study it is concluded that the test item is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.