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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

Constituent 1
Chemical structure
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 resultsopen allclose all
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
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.