Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

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:

25 January 2018 - 07 December 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

EC Number:

948-518-4

Molecular formula:

(C13H23N3O2 . C31H43N3O6)x

IUPAC Name:

Reaction products of 2,3-epoxypropyl phenyl ether and 2,2'-iminodi(ethylamine)

Test material form:

liquid

Details on test material:

PGE-DETA adduct

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Olin Corportation Batch: 17-09-502-04 QM 14K17-54
- Expiration date of the lot/batch: 30 October 2019
- Purity test date: Approx. 97% (including the oligomeric isomers)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: All formulations were prepared freshly before treatment and used within two hours of preparation. The formulation was assumed to be stable for this period.

Test Item Preparation
On the day of the experiment, the test item 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether was suspended in Ethanol (purity > 99 %). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. The test item was neutralized with HCL 2M.

Method

Target gene:

Histidine locus of several Salmonella typhimurium strains
Tryptophan locus of Escherichia coli WP2 uvrA

Species / strain

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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

In the pre-experiment (Experiment 1) the concentration range of the test item was 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate.
Since strong toxic effects were observed in experiment I, a minimum of six concentration was tested in experiment II.

Experiment II: Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; and 1000 μg/plate;
Strain, TA 100: 0.3; 1, 3, 10; 33; 100; and 333 μg/plate; Strain WP2 uvrA: 10; 33; 100; 333; 1000; and 2500 μg/plate;
Based on the observed toxicity in the different strains 2500, 1000 or 333 μg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. Since the acceptance criterion of at least five analysable dose levels was not met in strain TA 98 and WP2 uvrA, both without metabolic activation, this part of experiment II was repeated as a pre-incubation assay with the following concentrations: 0.3; 1; 3, 10; 33; 100; 333; and 1000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility properties and its relative nontoxicity to the bacteria.

- Justification for percentage of solvent in the final culture medium:

Controls

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 (TA 1537, TA 98 w/o S9)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10^8-10^9 cells/mL
- Test substance in vehicle added in agar (plate incorporation) or to suspenstion (pre-incubation);

TREATMENT AND HARVEST SCHEDULE:
Plate Incorporation: After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.
- Preincubation period: Suspension incubated at 37°C for 60 minutes after addition of Test Article. After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Rationale for test conditions:

Though the plate incorporation method was clearly negative, the pre-incubation method was also evaluted and was clearly negative.

Evaluation criteria:

Assay Acceptability 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 twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (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 Criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twice or above (strains TA 98, TA 100, and WP2 uvrA) or of thrice 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 μg/plate without S9 mix and from 1000 to 5000 μg/plate with S9 mix, in experiment II from 1000 μg/plate up to the highest investigated dose in all strains with S9 mix and in experiment IIa at 1000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 1000 to 5000 μg/plate with and without S9 mix and in experiment II and IIa from 333 up to the highest investigated dose with (experiment II) and without S9 mix (experiment II and IIa). The undissolved particles had no influence on the data recording.

- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES:
Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. 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): TA 1535, and 1537 at 1000 ug/plate and above with and w/o S9, TA 98 at 1000 ug/plate and above w/o S9 and at 333 ug/plate with S9; TA 100 at 100 ug/plate and above w/o S9 and at 333 ug/plate and above with S9, WP2 at 2500 and above with and w/o S9.

Pre-incubation (Experiment II/IIa):
Experiment II:
Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; and 1000 μg/plate
Strain, TA 100: 0.3; 1, 3, 10; 33; 100; and 333 μg/plate
Strain WP2 uvrA: 10; 33; 100; 333; 1000; and 2500 μg/plate
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. 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): TA 1535, and 1537 at 333 ug/plate and above w/o S9 and at 1000 ug/plate with S9. , TA 98 at 333 ug/plate and above w and w/o S9; TA 100 at 100 ug/plate and above w/o S9 and at 333 ug/plate and above with S9, WP2 at 100 ug/plate and above w/o S9 and at 333 and above with and w/o S9, WP2 at 333 ug/plate w/o S9 and at 2500 and above with and w S9.

Experiment IIa:
Strains TA 98 and WP2 uvrA: 0.3; 1; 3, 10; 33; 100; 333; and 1000 μg/plate
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. 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): TA 98 at 100 ug/plate and above w/o S9; WP2 at 333 ug/plate and above w/o S9.

Historical Control data are included as an Annex of the study report.


STUDY RESULTS
- Concurrent vehicle negative and positive control data

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
- Statistical analysis; p-value if any
- Any other criteria: e.g. GEF for MLA

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

Any other information on results incl. tables

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	1000 – 5000	1000 – 5000	333 – 1000	1000	n.p.
TA 1537	1000 – 5000	1000 – 5000	333 – 1000	1000	n.p.
TA 98	1000 – 5000	333 – 5000	333 – 1000	333 – 1000	100 – 1000
TA 100	100 – 5000	333 – 5000	100 – 333	333	n.p.
WP2 uvrA	2500 – 5000	2500 – 5000	333 – 2500	2500	333 - 1000

n.p. = not performed

Table 1 Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Ethanol		10 ± 5	8 ± 4	29 ± 6	155 ± 6	33 ± 3
	Untreated		11 ± 5	10 ± 1	34 ± 4	176 ± 14	42 ± 2
	1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether	3 μg	9 ± 2	8 ± 3	34 ± 8	166 ± 14	32 ± 4
		10 μg	10 ± 4	11 ± 1	30 ± 4	155 ± 15	31 ± 5
		33 μg	14 ± 5	8 ± 3	32 ± 4	102 ± 20	28 ± 8
		100 μg	8 ± 2	9 ± 2	18 ± 4	16 ± 5	30 ± 6
		333 μg	5 ± 1	6 ± 1	13 ± 3	3 ± 2	30 ± 4
		1000 μg	1 ± 1 P	1 ± 1P M	2 ± 1P M	0 ± 0 P	15 ± 4M P
		2500 μg	0 ± 0 P	0 ± 0P M	0 ± 1P M	0 ± 0 P	0 ± 1M P
		5000 μg	0 ± 0 P	0 ± 0P M	0 ± 0P M	0 ± 0 P	0 ± 0M P
	NaN3	10 μg	915 ± 19			1824 ± 53
	4-NOPD	10 μg			310 ± 4
		50 μg		95 ± 5
	MMS	2.0 μL					831 ± 51
With Activation	Ethanol		13 ± 5	9 ± 4	45 ± 5	135 ± 11	35 ± 12
	Untreated		10 ± 4	10 ± 4	10 ± 4	10 ± 4	10 ± 4
	1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether	3 μg	13 ± 3	11 ± 1	46 ± 13	122 ± 12	34 ± 8
		10 μg	12 ± 3	12 ± 3	39 ± 5	160 ± 4	33 ± 6
		33 μg	13 ± 3	8 ± 2	45 ± 6	130 ± 5	39 ± 11
		100 μg	10 ± 3	8 ± 2	37 ± 1	89 ± 14	34 ± 4
		333 μg	13 ± 2	8 ± 2	15 ± 3	18 ± 3	33 ± 8
		1000 μg	4 ± 2P M	2 ± 1P M	6 ± 1P M	0 ± 1P M	21 ± 2 P
		2500 μg	0 ± 1P M	0 ± 1P M	0 ± 1P M	0 ± 0P M	7 ± 2 P
		5000 μg	0 ± 0P M	0 ± 0P M	0 ± 0P M	0 ± 0P M	0 ± 1 P
	2-AA	2.5 μg	355 ± 43	127 ± 12	3692 ± 513	2585 ± 156
		10 μg					365 ± 19

Key to Positive Controls                                   Key to Plate Postfix Codes

NaN3 sodium azide                                          P Precipitate

2-AA 2-aminoanthracene                                  M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

Table 2 Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Ethanol		10 ± 0	9 ± 2	31 ± 3	194 ± 26	29 ± 7
	Untreated		10 ± 3	9 ± 5	31 ± 6	214 ± 15	30 ± 4
	1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether	0.3 μg				180 ± 26
		1 μg				170 ± 10
		3 μg	7 ± 2	10 ± 4	32 ± 8	194 ± 9
		10 μg	10 ± 2	11 ± 5	35 ± 2	167 ± 6	30 ± 3
		33 μg	10 ± 3	10 ± 3	33 ± 2	94 ± 11	27 ± 3
		100 μg	6 ± 1	7 ± 2	18 ± 4	10 ± 2	30 ± 3
		333 μg	1 ± 1 P	1 ± 1 P	0 ± 0 P	0 ± 0 P	10 ± 3P M
		1000 μg	0 ± 0 P	0 ± 0 P	0 ± 0 P		0 ± 1P M
		2500 μg					0 ± 0P M
		5000 μg	0 ± 0 P	0 ± 0P M	0 ± 0P M	0 ± 0 P	0 ± 0M P
	NaN3	10 μg	1387 ± 46			2588 ± 128
	4-NOPD	10 μg			429 ± 20
		50 μg		64 ± 9
	MMS	2.0 μL					783 ± 28
With Activation	Ethanol		10 ± 4	10 ± 3	42 ± 8	187 ± 14	37 ± 10
	Untreated		14 ± 5	13 ± 2	29 ± 5	196 ± 7	39 ± 10
		0.3 ug				187 ± 4
	1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether	1 ug				183 ± 18
		3 μg	12 ± 3	10 ± 1	47 ± 9	189 ± 8	34 ± 8
		10 μg	9 ± 2	8 ± 1	44 ± 3	179 ± 2	42 ± 5
		33 μg	10 ± 4	10 ± 2	49 ± 10	178 ± 18	39 ± 5
		100 μg	10 ± 2	10 ± 4	42 ± 5	127 ± 6	39 ± 5
		333 μg	12 ± 3 P	8 ± 4 P	11 ± 3 P	18 ± 4P M	32 ± 7 P
		1000 μg	4 ± 1P M	0 ± 0 P	1 ± 1 P		17 ± 3P M
		2500 μg					3 ± 1P M
	2-AA	2.5 μg	441 ± 16	129 ± 2	4627 ± 313	5126 ± 121
		10 μg					408 ± 41

Key to Positive Controls                                   Key to Plate Postfix Codes

NaN3 sodium azide                                          P Precipitate

2-AA 2-aminoanthracene                                  M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

Table 3 Summary of Experiment IIa

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 98	WP2 uvrA
Without Activation	Ethanol		30 ± 5	30 ± 4
	Untreated		23 ± 2	37 ± 4
	1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether	0.3 μg	28 ± 4	26 ± 5
		1 μg	21 ± 5	28 ± 4
		3 μg	25 ± 2	28 ± 7
		10 μg	21 ± 5	30 ± 7
		33 μg	23 ± 2	28 ± 6
		100 μg	13 ± 4	25 ± 7
		333 μg	0 ± 1 P	10 ± 4 P
		1000 μg	0 ± 0 P	0 ± 1 P
	4-NOPD	10 μg	513 ± 13
	MMS	2.0 μL		529 ± 69

Key to Positive Controls                                   Key to Plate Postfix Codes

NaN3 sodium azide                                          P Precipitate

2-AA 2-aminoanthracene                                  M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

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.
Therefore, 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II and IIa) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

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 liver microsomal activation (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:

Strains TA 1535, TA 1537, and TA 98: 3, 10; 33; 100; 333; and 1000 μg/plate

Strain, TA 100: 0.3; 1, 3, 10; 33; 100; and 333 μg/plate

Strain WP2 uvrA: 10; 33; 100; 333; 1000; and 2500 μg/plate

Experiment IIa:

Strains TA 98 and WP2 uvrA: 0.3; 1; 3, 10; 33; 100; 333; and 1000 μg/plate

The test item precipitated in the overlay agar in the test tubes in experiment I at 5000 μg/plate without S9 mix and from 1000 to 5000 μg/plate with S9 mix, in experiment II from 1000 μg/plate up to the highest investigated dose in all strains with S9 mix and in experiment IIa at 1000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed in experiment I from 1000 to 5000 μg/plate with and without S9 mix and in experiment II and IIa from 333 up to the highest investigated dose with (experiment II) and without S9 mix (experiment II and IIa). The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether 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.

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.

Therefore, 1,2-Ethanediamine, N-(2-aminoethyl)-, reaction products with glycidyl Ph ether is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.