Amines, polyethylenepoly-, tetraethylenepentamine fraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames tests and a Sister Chromatide Exchange test are available with Amines, polyethylenepoly-, tetraethylenepentamine fraction which showed both positive genotoxic potential.

An in vitro micronucleus test according to OECD 487 is ongoing and the draft results are negative. As soon as the final report is available the dossier will be updated. In the meanwhile the following interim data is used in addition. Reliable data from the structural analogue Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8) showed positive results in an vitro gene mutation (mammalian cells) test.

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:

12 August 2020 - 07 September 2020

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:

21 July 1997 as corrected in 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

August 1998

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines

Version / remarks:

31 March 2011

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) Federal Register

Version / remarks:

June 2012

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his / trp operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

other:

Remarks:

TA1537: his C 3076; rfa-; uvrB; TA98: his D 3052; rfa-; uvrB-; R-factor; TA1535: his G 46; rfa-; uvrB-; TA100: his G 46; rfa-; uvrB-; R-factor; WP2uvrA trp-; uvrA-;

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley);
- source of S9: purchased from Moltox; Lot No. 4222; the protein level was adjusted to 20 mg/mL;
- method of preparation of S9 mix: The S9-mix was prepared before using sterilized co-factors and maintained on ice for the duration of the test (S9: 5.0 mL, 1.65 M KCl/0.4 M MgCl2: 1.0 mL, 0.1 M glucose-6-phosphate: 2.5 mL, 0.1 M NADP: 2.0 mL, 0.2 M sodium phosphate buffer (pH 7.4): 25.0 mL, sterile distilled water 14.5 mL);
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (i.e. 0.05 mL S9)
- quality controls of S9: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of the experiment.

Test concentrations with justification for top dose:

- Experiment 1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (5000 µg/plate is the maximum recommended dose level according to OECD TG 471);
- Experiment 2 (preincubation): 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate
Dose range used for Experiment 2 was determined by the results of Experiment 1. Seven test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non-toxic dose levels as required by the test guideline, and were selected based on the lack of cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation. An intermediate dose level (3000 µg/plate) was also included following the observation of small but statistically significant increases in revertant colony frequency noted in the first mutation test.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle:
The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks.

The test item was accurately weighed and, on the day of the experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for test item purity with a correction factor of 1.02 employed. All test item preparation and dosing was performed under yellow safety lighting. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations was not determined.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 9-Aminoacridine hydrochloride monohydrate: -S9; in DMSO; TA1537: 80 µg/plate; 2-Aminoanthracene: +S9; in DMSO; TA100: 1 µg/plate; TA1537 / TA1535: 2 µg/plate; WP2uvrA: 10 µg/plate;

Remarks:

In addition, sterility controls (top agar and histidine / biotin or tryptophan -S9, top agar and histidine / biotin or tryptophan +S9, maximum dosing solution of the test item -S9) were performed in singular prior to mutation test.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate;
- Number of independent experiments: 2;

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation; Experiment 1); preincubation (Experiment 2);

Experiment 1:
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer or S9-mix and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.

Experiment 2:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer or S9-mix and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method (see above, Experiment 1).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period (Experiment 2): 20 minutes;
- Exposure duration/duration of treatment: between 48 and 72 hours;

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (the plates were viewed microscopically for evidence of thinning of the background bacterial lawn);


METHODS FOR MEASUREMENTS OF GENOTOXICIY: Plates were scored for the presence of revertant colonies using an automated colony counting system after incubation at 37 +/- 3 °C.

Rationale for test conditions:

according to OECD TG 471

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against historical control ranges of the lab.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response).
5. Statistical analysis of data as determined by UKEMS.

A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnett’s Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Experiment 1: Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate; Experiment 2: statistically significant, dose-related increases from 500 µg/plate -S9 and from 3000 µg/plate +S9;

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) and at / above 1500 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) +S9 and at / above 3000 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) +S9 and at / above 3000 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Experiment 2: Small but statistically significant increases in the frequency of revertant colonies at / above 3000 µg/plate -S9; individual revertant colony counts at the upper limit or in excess of the historical control maxima;

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks.
- Precipitation and time of the determination: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiment 1 and 2.
- Other confounding effects:
Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Results for the negative controls (spontaneous mutation rates) and viability were considered to be acceptable. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the historical control range of the lab in both the absence and presence of S9. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. All of the acceptability criteria were considered to be met. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Ames test:
- Signs of toxicity: There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix) in Experiment 1 and 2.
- Results:
Experiment 1 (plate incorporation): Small but statistically significant increases in the frequency of revertant colonies were recorded with doses of the test item at 5000 µg/plate for bacterial strains TA100 and WP2uvrA (absence and presence of S9) and TA1535 and TA98 in the presence of S9. Whilst a two or three-fold increase was not achieved for any of the bacterial strains, individual revertant colony counts were at the upper limit or in excess of the historical untreated/vehicle control maxima of the lab.

Experiment 2 (preincubation): Statistically significant and dose-related increases in WP2uvrA revertant colony frequency were noted from 500 µg/plate in the absence of S9 and 3000 µg/plate in the presence of S9. Maximum increases in excess of two-fold (of 6.3-fold and 3.6-fold) when compared to the concurrent vehicle controls were noted in the absence and presence of S9 respectively. These responses were also accompanied by individual colony counts in excess of the historical untreated/vehicle control maxima of the lab for the strain with clear evidence of dose-related increase. There were also smaller statistically significant increases noted in TA100, TA1535, TA98 (absence and presence of S9) and TA1537 in the absence of S9 at and/or above 1500 µg/plate. Whilst these increases did not achieve a 2 or 3-fold response (depending on tester strain type) over the concurrent vehicle control, the individual revertant colony counts, particularly in the case of TA100 dosed in the presence of S9, were in excess of the in-house
historical control maxima.

See Tables 1 - 4 under Any other information on results incl. tables for individual plate counts & mean number of revertant colonies per plate and standard deviation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and the number of data)
- Positive historical control data (2018):
TA100, -S9: 220 - 1525, mean 606, SD 213.6, n = 306
TA100, +S9: 422 - 3928, mean 1726, SD 528.7, n = 300
TA1535, -S9: 74 - 2601, mean 653, SD 484.4, n = 272
TA1535, +S9: 113 - 481, mean 301, SD 57.2, n = 271
WP2uvrA, -S9: 111 - 1420, mean 706, SD 235.8, n = 254
WP2uvrA, +S9: 105 - 697, mean 230, SD 74.8, n = 251
TA98, -S9: 97 - 461, mean 212, SD 77.1, n = 292
TA98, +S9: 79 - 342, mean 158, SD 49.3, n = 292
TA1537, -S9: 86 - 833, mean 274, SD 150.4, n = 276
TA1535, +S9: 116 - 541, mean 294, SD 86.8, n = 272
- Positive historical control data (2019):
TA100, -S9: 205 - 2322, mean 622, SD 294.0, n = 239
TA100, +S9: 318 - 2561, mean 1381, SD 442.8, n = 234
TA1535, -S9: 69 - 4595, mean 790, SD 825.3, n = 230
TA1535, +S9: 112 - 1976, mean 268, SD 124.0, n = 230
WP2uvrA, -S9: 117 - 1391, mean 629, SD 245.6, n = 204
WP2uvrA, +S9: 99 - 790, mean 175, SD 70.8, n = 202
TA98, -S9: 92 - 477, mean 186, SD 71.4, n = 253
TA98, +S9: 88 - 719, mean 165, SD 75.5, n = 246
TA1537, -S9: 76 - 830, mean 266, SD 142.4, n = 230
TA1535, +S9: 109 - 1964, mean 232, SD 127.9, n = 224
- Negative (combined vehicle / untreated) historical control data (2018):
TA100, -S9: 67 - 170, mean 122, SD 18.8, n = 301
TA100, +S9: 64 - 187, mean 125, SD 21.5, n = 297
TA1535, -S9: 7 - 33, mean 17, SD 4.2, n = 542
TA1535, +S9: 9 - 28, mean 14, SD 3.1, n = 279
WP2uvrA, -S9: 11 - 44, mean 27, SD 5.3, n = 511
WP2uvrA, +S9: 20 - 53, mean 36, SD 6.2, n = 253
TA98, -S9: 11 - 41, mean 22, SD 4.5, n = 583
TA98, +S9: 15 - 50, mean 27, SD 5.1, n = 300
TA1537, -S9: 5 - 25, mean 12, SD 3.3, n = 550
TA1535, +S9: 3 - 22, mean 13, SD 3.2, n = 280
- Negative (combined vehicle / untreated) historical control data (2019):
TA100, -S9: 75 - 168, mean 116, SD 18.1, n = 240
TA100, +S9: 81 - 181, mean 122, SD 18.8, n = 234
TA1535, -S9: 9 - 38, mean 17, SD 4.8, n = 462
TA1535, +S9: 7 - 31, mean 14, SD 3.6, n = 237
WP2uvrA, -S9: 12 - 47, mean 25, SD 5.9, n = 410
WP2uvrA, +S9: 16 - 55, mean 32, SD 6.7, n = 205
TA98, -S9: 12 - 39, mean 23, SD 5.4, n = 508
TA98, +S9: 13 - 46, mean 28, SD 5.9, n = 249
TA1537, -S9: 4 - 25, mean 12, SD 3.4, n = 462
TA1535, +S9: 6 - 25, mean 13, SD 3.3, n = 227

Table 1: Results of Experiment 1 (plate incorporation) - without metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	128 132 115	(125) 8.9#	37 22 21	(27) 9.0	26 26 27	(26) 0.6	25 21 27	(24) 3.1	13 9 14	(12) 2.6
1.5 µg	142 115 139	(132) 14.8	27 24 18	(23) 4.6	16 13 32	(20) 10.2	24 25 27	(25) 1.5	13 17 12	(14) 2.6
5 µg	140 110 133	(128) 15.7	28 18 24	(23) 5.0	20 19 35	(25) 9.0	28 16 27	(24) 6.7	10 13 13	(12) 1.7
15 µg	127 140 123	(130) 8.9	34 39 19	(31) 10.4	21 29 23	(24) 4.2	23 28 22	(24) 3.2	17 11 12	(13) 3.2
50 µg	134 133 110	(126) 13.6	32 34 25	(30) 4.7	22 19 23	(21) 2.1	33 28 28	(30) 2.9	12 11 12	(12) 0.6
150 µg	159 114 130	(134) 22.8	23 38 18	(26) 10.4	28 25 22	(25) 3.0	29 17 37	(28) 10.1	10 12 14	(12) 2.0
500 µg	125 126 122	(124) 2.1	18 15 28	(20) 6.8	29 30 16	(25) 7.8	25 13 24	(21) 6.7	9 23 13	(15) 7.2
1500 µg	126 131 137	(131) 5.5	29 35 29	(31) 3.5	35 41 21	(32) 10.3	31 15 27	(24) 8.3	8 18 8	(11) 5.8
5000 µg	175 179 168	(174) 5.6 ***	36 35 38	(36) 1.5	46 46 43	(45) 1.7 *	30 36 41	(36) 5.5	16 16 11	(14) 2.9
Positive controls -S9
Name	ENNG		ENNG		ENNG		4NQO		9AA
Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
No. of Revertants	651 691 703	(682) 27.2	405 514 456	(458) 54.5	745 732 669	(715) 40.6	115 107 101	(108) 7.0	263 452 232	(316) 119.1

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

* p≤0.05

*** p≤0.001

# Standard deviation

Table 2: Results of Experiment 1 (plate incorporation) - with metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	126 138 139	(134) 7.2#	15 23 13	(17) 5.3	29 30 22	(27) 4.4	30 30 25	(28) 2.9	11 10 13	(11) 1.5
1.5 µg	128 127 137	(131) 5.5	18 11 12	(14) 3.8	24 29 22	(25) 3.6	32 35 35	(34) 1.7	9 12 6	(9) 3.0
5 µg	132 132 138	(134) 3.5	16 13 19	(16) 3.0	21 23 31	(25) 5.3	27 30 33	(30) 3.0	9 9 10	(9) 0.6
15 µg	105 121 148	(125) 21.7	19 26 17	(21) 4.7	20 27 16	(21) 5.6	40 34 29	(34) 5.5	10 9 9	(9) 0.6
50 µg	105 115 96	(105) 9.5	17 17 7	(14) 5.8	23 23 33	(26) 1.7	31 31 39	(34) 4.6	8 12 6	(9) 3.1
150 µg	131 111 137	(126) 13.6	19 13 25	(19) 6.0	28 25 25	(26) 1.7	31 31 39	(34) 4.6	8 14 7	(10) 3.8
500 µg	140 120 161	(140) 20.5	12 19 29	(20) 8.5	23 20 38	(27) 9.6	26 35 34	(32) 4.9	8 12 6	(9) 3.1
1500 µg	142 149 131	(141) 9.1	30 20 21	(24) 5.5	34 29 25	(29) 4.5	29 32 32	(31) 1.7	13 5 12	(10) 4.4
5000 µg	229 208 218	(218) 10.5 ***	21 35 41	(32) 10.3 *	43 48 38	(43) 5.0 *	49 45 46	(47) 2.1 ***	18 14 14	(15) 2.3
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1528 1582 1782	(1631) 133.8	278 267 288	(278) 10.5	154 178 181	(171) 14.8	228 221 226	(225) 3.6	303 256 247	(269) 30.1

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

*** p≤0.001

# Standard deviation

Table 3: Results of Experiment 2 (preincubation) - without metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	144 145 148	(146) 2.1#	18 15 21	(18) 3.0	15 20 18	(18) 2.5	18 20 18	(19) 1.2	7 14 10	(10) 3.5
15 µg	140 142 138	(140) 2.0	21 16 15	(17) 3.2	13 22 17	(17) 4.5	19 19 23	(20) 2.3	10 14 10	(11) 2.3
50 µg	106 111 135	(117) 15.5	15 16 16	(16) 0.6	15 22 18	(18) 3.5	20 17 24	(20) 3.5	11 14 12	(12) 1.5
150 µg	122 132 129	(128) 5.1	12 12 12	(12) 0.0	19 15 20	(18) 2.6	23 21 23	(22) 1.2	8 9 9	(9) 0.6
500 µg	143 163 145	(150) 11.0	16 19 16	(17) 1.7	36 27 31	(31) 4.5 *	16 15 14	(15) 1.0	8 6 7	(7) 1.0
1500 µg	148 143 140	(144) 4.0	24 16 12	(17) 6.1	36 42 57	(45) 10.8 ***	17 26 18	(20) 4.9	7 8 9	(8) 1.0
3000 µg	143 173 139	(152) 18.6	22 25 32	(26) 5.1 *	61 43 68	(57) 12.9 ***	42 24 40	(35) 9.9 ***	25 14 19	(19) 5.5 **
5000 µg	188 179 178	(182) 5.5 **	28 22 30	(27) 4.2 *	123 107 110	(113) 8.5 ***	31 35 38	(35) 3.5 ***	21 26 24	(24) 2.5 ***
Positive controls -S9
Name	ENNG		ENNG		ENNG		4NQO		9AA
Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
No. of Revertants	879 931 585	(798) 186.6	519 475 665	(553) 99.5	843 844 796	(828) 27.4	238 216 219	(224) 11.9	342 317 346	(335) 15.7

ENNG:N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

* p≤0.05

** p≤0.01

*** p≤0.001

# Standard deviation

Table 4: Results of Experiment 2 (preincubation) - with metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	147 144 132	(141) 7.9#	15 19 19	(18) 2.3	33 17 27	(26) 8.1	25 26 36	(29) 6.1	13 5 16	(11) 5.7
15 µg	141 145 144	(143) 2.1	13 8 17	(13) 4.5	29 20 34	(28) 7.1	34 20 28	(27) 7.0	12 8 12	(11) 2.3
50 µg	134 138 126	(133) 6.1	26 11 17	(18) 7.5	23 26 20	(23) 3.0	32 22 25	(26) 5.1	8 15 11	(11) 3.5
150 µg	157 141 132	(143) 12.7	15 8 9	(11) 3.8	25 29 21	(25) 4.0	16 27 31	(25) 7.8	10 11 13	(11) 1.5
500 µg	165 142 149	(152) 11.8	14 7 13	(11) 3.8	30 28 29	(29) 1.0	30 38 32	(33) 4.2	10 18 6	(11) 6.1
1500 µg	196 178 192	(189) 9.5 ***	15 9 18	(14) 4.6	42 46 28	(39) 9.5	37 22 35	(31) 8.1	10 7 15	(11) 4.0
3000 µg	248 246 218	(237) 16.8 ***	26 29 31	(29) 2.5	44 49 53	(49) 4.5 **	41 31 38	(37) 5.1	12 13 12	(12) 0.6
5000 µg	250 257 267	(258) 8.5	32 35 46	(38) 7.4 **	88 109 83	(93) 13.8 ***	59 57 54	(57) 2.5 ***	38 9 23	(23) 14.5
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1269 1194 1146	(1203) 62.0	223 235 233	(230) 6.4	134 144 163	(147) 14.7	130 126 533	(263) 233.8	238 249 220	(236) 14.6

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

** p≤0.01

*** p≤0.001

# Standard deviation

Conclusions:

positive with and without metabolic activation

Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was positive in the Ames assay under the conditions and according to the criteria of the test protocol.

Executive summary:

In the reverse mutation assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli according to OECD TG 471 the test item, Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) met the criteria for a positive result, both with and without metabolic activation (S9-mix). Under the conditions of this test Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was considered to be mutagenic

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

DRAFT results

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2020 - 03 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

24-hour exposure: incubation for further 24 h with Cytochalasin B after the 24 h treatment

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphocytes
- Suitability of cells: Human peripheral blood lymphocytes are recognized in the OECD 487 guideline as being a suitable cell line for the in vitro Mammalian Cell Micronucleus Test.

- Normal cell cycle time (negative control): Average generation time (AGT) for human lymphocytes is considered to be approximately 16 hours; therefore, using this average the exposure time for the experiments for 1.5 x AGT is 24 hours;

For lymphocytes:
- Sex, age and number of blood donors: non smoking volunteer (18 - 35 years of age), who had been previously screened for suitability (had not knowingly been exposed to high levels of radiation or hazardous chemicals, had not knowingly recently suffered from a viral infection); details of the donors: Preliminary Toxicity Test: female, aged 35 years; Main Experiment: female, aged 34 years;
- Whether whole blood or separated lymphocytes were used: For each experiment, sufficient whole blood was drawn from the peripheral circulation.
- Whether blood from different donors were pooled or not: no different donors;
- Mitogen used for lymphocytes: phytohaemagglutinin (PHA);

MEDIA USED
- Culture conditions: Cells (whole blood cultures) were grown in Eagle's minimal essential medium (MEM) with HEPES buffer (MEM), supplemented with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

yes (Cytochalasin B at a final concentration of 4.5 µg/mL)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley);
- source of S9: purchased from Moltox; Lot No. 4222 (expiry date of 12 March 2022); the protein level was adjusted to 20 mg/mL;
- method of preparation of S9 mix: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5 mM).
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
- quality controls of S9: The S9 was pre-tested for acceptability by the supplier prior to purchase and was supplied with a relevant “Quality Control & Production Certificate”.

Test concentrations with justification for top dose:

- 4 h / -S9: 0*, 29.30, 58.59, 117.19, 234.38*, 468.75*, 937.5* µg/mL
- 4 h / +S9: 0*, 29.30, 58.59, 117.19, 234.38*, 468.75*, 937.5* µg/mL
- 24 h / -S9: 0*, 7.33, 14.65*, 29.30*, 43.95*, 58.59, 87.89, 117.19 µg/mL

(*Dose levels selected for evaluation of micronucleus frequency in binucleate cells)

The test item was a multi-constituent substance and therefore the maximum recommended dose according to OECD TG 487was initially set at 5000 µg/mL The osmolality did not increase by more than 50 mOsm when the test item was dosed into media. However, marked increases in pH of greater than 1 pH unit were observed at and above 1250 µg/mL when the test item was dosed into media and, therefore, the maximum concentration tested was limited to 937.5 µg/mL due to pH (see also Table 1 under Any other information on results incl. tables for the results of the pH and osmolality readings). This is considered to be in compliance with the OECD TG 487, which states that concentrations which have the capability of producing artefactual results such as a marked change in pH should be avoided.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium (MEM)

- Justification for choice of solvent/vehicle:
The test item was miscible in Minimal Essential Medium (MEM) at 50 mg/mL in solubility checks. MEM was therefore selected as the preferred solvent.

Prior to each experiment, the test item was accurately weighed, formulated in MEM medium, and serial dilutions prepared. A correction for the purity of the test item of 97.7% was applied to the formulations.The test item was formulated within two hours of it being applied to the test system; it is assumed that the test item formulation was stable for this duration. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

MEM

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine / -S9: 0.075 µg/mL in sterile distilled water for 24-hour exposure

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate lymphocyte cultures (A and B), (quadruplicate for the solvent) were established for each dose level;
- Number of independent experiments: 1

METHOD OF TREATMENT/ EXPOSURE:
Lymphocyte cultures were established for each dose level, by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.25 - 9.30 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.50 - 0.55 mL heparinized whole blood
- 4-hour exposure: After approximately 44 - 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of solvent control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. For the experiments with metabolic activation, 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. The nominal total volume of each culture was 10 mL All cultures were then returned to the incubator.
- 24-hour exposure: The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 44 - 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of solvent control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4 h (- / +S9), 24 h
- Harvest time after the end of treatment (sampling/recovery times):
- 4-hour exposure: After the exosure at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 µg/mL, and then incubated for a further 24 hours before the harvest of the cells.

- 24-hour exposure: After exposure for 24 h, the tubes and the cells were washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 µg/mL, and then the cells were incubated for a further 24 hours before the harvest of the cells. The extended exposure detailed above is a modification of the suggested cell treatment schedule in the OECD TG 487 and is considered to be an acceptable alternative. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item was unknown prior to the start of the study this modification of the schedule was considered more effective and reproducible due to the observations on human lymphocytes in the lab and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.

At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375 M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry with gentle warming. Each slide was permanently labeled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 binucleated cells were analyzed per culture (2000 binucleated cells per concentration for the test item and positive control and 4000 binucleated cells for the solvent controls);
- Criteria for scoring micronucleated cells: Cells with 1, 2 or more micronuclei were recorded and included in the total; the criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nucleii; binucleate cells were selected for scoring, if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary; the two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter;

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity:
A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the solvent controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPI(T) – 1) / (CBPI(C) – 1)}
Where:
CBPI = (No. mononucleated cells + (2 x No. binucleate cells) + (3 x No. multinucleate cells)) / Total number of cells


(T) = test chemical treatment culture
(C) = solvent control culture

Rationale for test conditions:

according to OECD TG 487 and modified when regarded as necessary

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase when evaluated with an appropriate trend test.
3. The results in all evaluated dose groups are within the range of the laboratory historical control data.
The test item is then considered to be unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. The increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.

There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions could be useful.

Statistics:

The frequency of binucleate cells with micronuclei was compared, with the concurrent solvent control value using the Chi-squared Test on observed numbers of cells with micronuclei. A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei.
The dose-relationship (trend-test) was assessed using a linear regression model. An arcsin square-root transformation was applied to the percentage of binucleated cells containing micronuclei (excluding positive controls). A linear regression model was then applied to these transformed values with dose values fitted as the explanatory variable. The F-value from the model was assessed at the 5% statistical significance level.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 24-hour exposure group in the absence of S9 only; in the 4-hour exposure groups with and without S9 the maximum concentration was limited due to marked increases in pH of greater than 1 at and above 1250 µg/mL;

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Marked increases in pH of greater than 1 pH unit were observed at and above 1250 µg/mL and, therefore, the maximum concentration was limited to 937.5 µg/mL due to pH for the 4-h exposure duration groups with and without metabolic activation (please see also Table 1 under Any other information on results incl. tables for further details). This is considered to be in compliance with the OECD 487 Guideline which states that concentrations which have the capability of producing artefactual results such as a marked change in pH should be avoided.
- Data on osmolality: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media (please see also Table 1 under Any other information on results incl. tables for further details).
- Water solubility: The test item was miscible in Minimal Essential Medium (MEM) at 50 mg/mL in solubility checks. MEM was therefore selected as the preferred solvent. Thus, the test substance is regarded as water soluble.
- Precipitation and time of the determination: No precipitate of the test item was observed either in the preliminary toxicity test or in the main experiment.

RANGE-FINDING/SCREENING STUDIES:
The preliminary toxicity test was performed using the exposure conditions as described for the main experiment but using single cultures for the test item dose levels and duplicate cultures for the solvent controls. The used exposure groups are the same as described for the main experiment (4-hour exposure - / +S9 & 24-hour exposure -S9; all followed by a 24-hour incubation period with treatment-free media, in the presence of CytB, prior to the cell harvest). The dose levels used were 0, 3.66, 7.32, 14.65, 29.30, 58.59, 117.19, 234.38, 468.75, and 937.5 µg/mL (the maximum dose was considered to be the maximum practical dose level due to marked increases in pH). Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods. Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the exposure groups of the main experiment. Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present up to 937.5 µg/mL in all three of the exposure groups. The test item induced evidence of modest toxicity in the 24-hour exposure group only (>50% cytostasis at 58.59 µg/mL and above). The dose levels used in the main experiment were selected using data from this preliminary toxicity test where the results indicated that the maximum concentration should be the maximum practical concentration, i.e. 937.5 µg/mL, due to increases in pH in the 4-hour exposure groups with and without metabolic activation, and limited by test item-induced toxicity in the 24-hour exposure group in the absence of S9, i.e. 117.19 µg/mL No hemolysis was observed

STUDY RESULTS
(DRAFT results; please see also Table 2 under Any other information on results incl. tables for further details).
- Concurrent vehicle negative and positive control data: The solvent control cultures had frequencies of cells with micronuclei within the expected range and were considered acceptable for addition to the laboratory historical negative control data range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that were compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
The qualitative assessment of the slides determined that the toxicity observed in the 24-hour exposure group was similar to that observed in the preliminary toxicity test and that there were binucleate cells suitable for scoring at the maximum dose level in all three of the exposure groups. The CBPI data confirm the qualitative observations that no dose-related toxicity was observed in the 4-hour exposure groups in the absence or presence of S9. In the 24-hour exposure group in the absence of S9, dose-related toxicity was observed and optimum toxicity was achieved with cytostasis in the required 55 ± 5% range at 43.95 µg/mL (59%). The maximum dose level selected for scoring of micronuclei in the binucleate cells was therefore 937.5 µgmL in the 4-hour exposure groups in both the absence and presence of S9 and 43.95 µg/mLin the 24-hour exposure group in the absence of S9.

- Genotoxicity results: In the 4-hour and 24-hour exposure groups in the absence of S9, the test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, all values were within the 95% control limits of the historical control data, and no concentration-dependent trend was observed. In the 4-hour exposure group in the presence of S9, a statistically significant increase in the frequency of binucleate cells with micronuclei was observed at 468.75 µg/mL. However, all values were within the 95% control limits of the historical control data, and no concentration-dependent trend was observed when evaluated with a trend test. The response was therefore considered to be spurious and of no toxicological significance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data (% binucleated cells with micronuclei):
- 4 h / -S9 (MMC): 1.75 - 6.80; mean: 3.71; SD: 1.25; 95% control limits: 1.21 - 6.21; n = 40;
- 4 h / +S9: 1.30 - 3.90; mean: 2.18; SD: 0.61; 95% control limits: 0.96 - 3.40; n = 40;
- 24 h / -S9: 2.00 - 10.65; mean: 4.31; SD: 1.75; 95% control limits: 0.81 - 7.81; n = 40;
- Negative (solvent/vehicle) historical control data (% binucleated cells with micronuclei):
- 4 h / -S9: 0.13 - 0.78; mean: 0.40; SD: 0.16; 95% control limits: 0.08 - 0.72; n = 40;
- 4 h / +S9: 0.13 - 1.05; mean: 0.39; SD: 0.21; 95% control limits: 0.00 - 0.81; n = 40;
- 24 h / -S9: 0.03 - 1.00; mean: 0.35; SD: 0.17; 95% control limits: 0.01 - 0.69; n = 40;

Remarks on result:

other: DRAFT results

Table 1: pH and osmolality readings

Dose Level (µg/mL)	0	19.53	39.06	78.13	156.25	312.5	625	937.5	1250	2500	5000
pH	7.28	7.30	7.32	7.37	7.44	7.57	7.78	8.03	8.35	9.14	9.6
Osmolality	320	324	330	323	322	321	322	323	323	326	338

Table 2: Results of the main experiment

Exposure Condition	Treatment/ Concentration (μg/mL)	Replicate	CBPI	Mean CBPI	Mean Cytostasis (%)	Binucleated cells containing micronuclei^a
						%	Mean	p-value^b	Trend testp-value^c
4-hour -S9	Vehicle (MEM)	A1 A2 B1 B2	1.46 1.47 1.45 1.54	1.48	0	1.30 0.30 0.90 0.40	0.73	/	0.314
	117.19	A B	1.40 1.41	1.41	16	/	/	/
	234.38	A B	1.41 1.47	1.44	8	0.60 0.20	0.40	/
	468.75	A B	1.42 1.41	1.42	14	0.80 0.40	0.60	/
	937.5	A B	1.46 1.44	1.45	6	0.20 0.50	0.35	/
	MMC 0.2	A B	1.26 1.37	1.32	34	2.40 3.50	2.95	1.396E-11***	/
4-hour +S9	Vehicle (MEM)	A1 A2 B1 B2	1.51 1.54 1.53 1.58	1.54	0	0.10 0.00 0.40 0.60	0.28	/	0.463
	117.19	A B	1.43 1.53	1.48	11	/	/	/
	234.38	A B	1.49 1.48	1.49	10	0.30 0.70	0.50	0.164
	468.75	A B	1.52 1.43	1.48	12	0.20 1.10	0.65	0.030*
	937.5	A B	1.42 1.45	1.44	19	0.20 0.60	0.40	/
	CP 7	A B	1.31 1.32	1.32	42	2.10 1.30	1.70	1.630E-09***	/
24-hour -S9	Vehicle (MEM)	A1 A2 B1 B2	1.96 1.92 2.06 2.15	2.02	0	0.80 0.20 0.70 0.30	0.50	/	0.124
	7.33	A B	1.78 1.82	1.80	22	/	/	/
	14.65	A B	1.78 1.73	1.76	26	0.30 0.70	0.50	/
	29.30	A B	1.57 1.52	1.55	47	0.90 0.60	0.75	/
	43.95	A B	1.45 1.39	1.42	59	1.10 0.60	0.85	0.1026
	58.59	A B	1.35 1.31	1.33	68	/	/	/
	87.89	A B	1.25 1.24	1.25	76	/	/	/
	117.19	A B	1.17 1.20	1.19	82	/	/	/
	DC 0.075	A B	1.45 1.37	1.41	60	4.20 4.10	4.15	1.059E-24***	/

MMC Mitomycin C

CP Cyclophosphamide

DC Demecolcine

MEM Minimal Essential Medium

^aThe percentage of micronucleated cells determined in a sample of 2000 binucleate cells (4000 for solvent)

^bp-values are for comparison with the control using Chi-square test

^cTrend test p-values using Linear regression model applied to control and test item concentrations

* P<0.05

*** P<0.001

Conclusions:

negative - DRAFT results

Executive summary:

The test item, Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA), did not induce any toxicologically significant increases in the frequency of binucleate cells with micronuclei in any of the three exposure groups, in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro under the described test conditions. Please consider, that the provided results are still draft results. Currently, there are further discussions ongoing with respect to the significant increases in pH. An update will be provided to the authority as soon as the final study report will be available.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without S9 >20 x 10E-2%

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Slesinski, 1981

Conclusions:

Interpretation of results: positive

The source substance Amines, polyethylenepoly-, triethylenetetramine fraction produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 80 x 10E-2% to 2.5 x 10E-2% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 10 x 10E-2% and 40 x 10E-2%. As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity.

Reference 4

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 3-6, 1987

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Version / remarks:

1986

Deviations:

no

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

SCE: interchanges between sister chromatids

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells are maintained in active growth by subculturing 2 to 3 times/week in antibiotic-free, Ham's Modified F12 Medium supplemented with 10% (v/v) heat-inactivated, fetal bovine sera (F12-10), and lacking in hypoxanthine.
For treatment of cells without metabolic activation, F12 medium with 50 units/ml of penicillin, 50 µg/mL streptomycin and 5% (v/v) of dialyzed bovine serum (F12-D5) is used.
For treatments incorporating an S9 metabolic activation system, identical medium, but without serum, is employed.
For determination of mutant frequencies, F12-D5 medium containing 2.0 µg/ml TG (6-thioguanine) is used as a "selective medium."
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

SCE:
-S9 mix 0.4, 0.6, 0.7, 0.8 mg/mL
+S9 mix 0.6, 0.8, 1.0, 2.0, 3.0 mg/mL

Test results from preliminary experiments with the test substance indicated that concentrations of 1.0 mg/mL or higher were excessively cytotoxic to CHO cells in the test without S9 activation and the treated cells were detached from the culture flask. With S9 acitivation, a slightly lower degree of growth inhibition was noted.

Vehicle / solvent:

cell culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

SCE Test - Production of SCEs following exposure to various concentrations of tetraethylenepentamine - sample A was studied with duplicate cultures of CHO cells tested both with and without the incorporation of a rat-liver S9 metabolic activation system. Various concentrations of tetraethylenepentamine - sample A for testing were attained by direct addition of various aliquots of the diluted test agent into the culture medium. Cell-culture medium was used as the diluent. All dilutions were prepared immediately prior to testing. For determination of direct mutagenic action, CHO cells were exposed to tetraethylenepentamine - sample A and appropriate controls for 5 hours without S9 activation. Indirect mutagenic action, requiring metabolic activation by liver S9 homogenate, was studied with a 2-hour exposure period. Bromodeoxyuridine (BrdU), required to differentiate between the individual sister chromatids by SCE staining, was present at a concentration of 3 µg/mL in the growth medium during treatment and during the culture period following exposure. A total of twenty-five cells/concentration was examined for SCE frequencies using duplicate cultures. At least 4 dose levels were tested both with and without metabolic activation. SCE production was determined for the highest 3 doses which did not produce excessive cytotoxic inhibition of cell division. The number of SCEs/cell, mean number of SCEs/chromosome and the level of statistical significance of the increases above the concurrent solvent control values are presented. The percentages of cells at various mitotic stages of cell division were monitored and recorded for indicating comparative cytotoxic effects.

Statistics:

Data from the SCE tests were analyzed by appropriate parametric tests following Standard Operating Procedures for statistical analyses at the Bushy Run Research Center.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the highest dose tested (0.8 mg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2 and 3 mg/mL dose grousps

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Statistically significant increases in the numbers of SCEs were observed with all of the doses of the test substance evaluated for SCE induction. The increases followed a dose-related trend and the 0.8 mg/mL high dose produced approximately a 1.8-fold increase above control values.

The number of SCEs produced by the concurrent EMS positive control was highly statistically different from the values for the concurrent solvent controls. These data indicated an appropriate sensitivity of the test system comparable to out historical positive control data. The number of SCEs obtained with the solvent and medium controls were also in an acceptable range of values included in the variability encountered in historical control values for this test.

Statistically significant increases in SCEs vere produced by all of the doses of the test agent evaluated. The quantitative increases, in SCEs showed a similar dose-related trend evident in the test without S9 activation, and SCE values attained approximately a 1.6-fold increase over concurrent controls.

The SCE values for the negative and solvent controls in the test with S9 activation were in an acceptable range of variability as encountered in previous experiments with this test system. Highly statistically significant numbers of SCEs were produced by the DMN positive control which indicated that the metabolic activation system was suitably active.

In the data from the test with S9, the proportion of 1st division cells increased commensurate with increasing test concentration. The 1.0 mg/mL dose produced approximately a 14% average decrease in the number of cells which completed two rounds of cell division in comparison to the control culture. In the test without S9 activation, no remarkable increases in 1st division cells were evident. The data from these two tests demonstrate that the doses evaluated for SCEs were in a biologically effective range of concentrations, but did not produce excessive cytotoxicity.

Conclusions:

Interpretation of results: positive

Sample A produced dose-related and statistically significant increases in the incidence of SCEs in the CHO cells exposed both in the presence and absence of a S9 metabolic activation system. Therefore, TEPA - Sample A was considered to produce a positive genotoxic effect in this test.

Executive summary:

Tetraethytenepentamine - Sample A was evaluated for potential genotoxic activity using the Sister Chromatid Exchange (SCE) test in Chinese hamster ovary (CHO) cells In vitro. The results indicated that the test chemical produced a dose-related, statistically significant genotoxic effect in tests conducted both with and without addition of a rat-liver S9 metabolic activation system. The quantitative SCE increases were approximately the same in the tests with and without metabolic activation, and were 1.8 and 1.6 above control, respectively. Therefore, TEPA - Sample A was considered to produce a positive genotoxic effect in this test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

There are no in vivo genetic toxicity studies available for Amines, polyethylenepoly-, tetraethylenepentamine fraction. Reliable data from the structural analogue Amines, polyethylenepoly-, triethylenetetramine fraction were used. In three in vivo Micronucleus Tests no genotoxic potential of the source substance were seen. In a Drosophila SLRL the source substance showed ambiguous results after feeding exposure and negative results after test substance injection.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Sex:

male

Genotoxicity:

ambiguous

Remarks:

after feeding exposure

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other:

Remarks:

Foureman, 1994

Sex:

male

Genotoxicity:

negative

Remarks:

after injection exposure

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks on result:

other:

Remarks:

Foureman, 1994

Conclusions:

Interpretation of results: negative
As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Remarks:

Summary of available data used for the endpoint assessment of the target substance

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

No. of animals per sex per dose:

5

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

No bone marrow toxicity was observed however at higher dose levels as administered in this test mortality occurs.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

Guzzie, 1987

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

SanSebastian, 1992

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other:

Remarks:

Foureman, 1994

Conclusions:

Interpretation of results: negative
Test results from several studies showed that the source substance Amines, polyethylenepoly-, triethylenetetramine fraction was not an active agent in producing treatment-related increases in micronuclei in male and female Swiss-Webster mice. Relatively high dosage levels of Amines, polyethylenepoly-, triethylenetetramine fraction were evaluated no treatment-related clastogenic activity was observed. Amines, polyethylenepoly-, triethylenetetramine fraction was considered to be inactive as a clastogenic agent in vivo under the conditions of those micronucleus tests. As explained in the analogue justification, it is considered that the target and the source substances are unlikely to lead to differences in genetic toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There are no data available to completely evaluate the genetic toxicity with Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640 -66 -7). In order to fulfil the standard information requirements set out in Annex VII-VIII, 8.4., in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from a structurally related substance was conducted. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across). Having regard to the general rules for grouping of substances and read-across approach laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 whereby substances may be predicted as similar provided that their physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8) is considered to be similar to Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7; TETA) on the basis of the structurally similar properties and/or activities. Therefore, the available genetic toxicity data on the source substance has been read-across to the target substance Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7). A detailed analogue approach justification is provided in the technical dossier (see IUCLID Section 13).

In vitro:

Gene mutation in bacteria:

Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7) was tested in concentrations up to 5000 µg/plate with and without metabolic activation for potential mutagenic activity using the bacterial reverse mutation assay (Ames test) according to OECD guideline 471 and under GLP conditions (Covance, 2021; RL1). In Experiment 1 (plate incorporation method), small but statistically significant increases in the frequency of revertant colonies were recorded with doses of the test item at 5000 µg/plate for bacterial strains S. typhimurium TA100 and E. coli WP2uvrA (absence and presence of S9) and S. typhimurium TA1535 and S. typhimurium TA98 in the presence of S9. Whilst a two or three-fold increase was not achieved for any of the bacterial strains, individual revertant colony counts were at the upper limit or in excess of the historical untreated/vehicle control maxima of the lab. In Experiment 2 (pre-incubation method), statistically significant and dose-related increases in E. coli WP2uvrA revertant colony frequency were noted from 500 µg/plate in the absence of S9 and 3000 µg/plate in the presence of S9. Maximum increases in excess of two-fold (of 6.3-fold and 3.6-fold) when compared to the concurrent vehicle controls were noted in the absence and presence of S9, respectively. These responses were also accompanied by individual colony counts in excess of the historical untreated/vehicle control maxima for the strain with clear evidence of dose-related increase. There were also smaller statistically significant increases noted in S. typhimurium strains TA100, TA1535, TA98 (absence and presence of S9) and TA1537 in the absence of S9 at and/or above 1500 µg/plate. Whilst these increases did not achieve a 2- or 3-fold response (depending on tester strain type) over the concurrent vehicle control, the individual revertant colony counts, particularly in the case of S. typhimurium TA100 dosed in the presence of S9, were in excess of the historical control maxima of the lab. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation and, no test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation. Taken together, Amines, polyethylenepoly-,tetraethylenepentamine fraction (TEPA)met the criteria for a positive result, both with and without metabolic activation (S9-mix). Under the conditions of this test Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was therefore considered to be mutagenic.

The positive outcome of the key study is supported by a second study, in which the registered substance Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was tested for potential mutagenic activity using the Salmonella/microsome bacterial mutagenicity assay (Ames test) similar to OECD guideline 471 (Willems, 1980). S. typhimurium strains TA100 and TA 1535 showed dose-dependent increase in revertant frequencies compared to control with and without metabolic activation. S. typhimurium strain TA1537 showed a dose-dependent increase in revertant frequency compared to control only with metabolic activation. Based on those results TEPA is considered to induce gene mutation in bacteria. No increase (compared to the negative control cultures) in revertant frequencies was seen in strain TA1538 and TA98. Taken together, TEPA was considered to be mutagenic in this in vitro bacterial assay.

In vitro cytogenicity:

Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7) was tested for its clastogenic and aneugenic potential using the in vitro micronucleus test according to OECD guideline 487 and under GLP conditions (Covance, 2021; RL1; DRAFT). Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells together with solvent and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B. The dose levels selected for the Main Experiment were as follows: 0*, 29.30, 58.59, 117.19, 234.38*, 468.75* and 937.5* µg/mL for the 4-hour exposure in the presence and absence of S9 and 0, 7.33, 14.65*, 29.30*, 43.95*, 58.59, 87.89, 117.19 µg/mL for the 24-hour exposure in the absence of S9 (*Dose levels selected for evaluation of micronucleus frequency in binucleate cells). The maximum concentration selected for the 4-hour exposure groups in the absence and presence of S9 was 937.5 µg/mL due to significant increases in pH of more than 1 pH unit at 1250 µg/mL and above. In addition, the maximum concentration selected for the 24-hour exposure group in the absence of S9 was 117.19 µg/mL due to test item-induced toxicity. The CBPI data indicated that no marked dose-related toxicity was observed in the 4-hour exposure groups in the absence or presence of S9, whereas in the 24-hour exposure group in the absence of S9, dose-related toxicity was observed (cytostasis > 50% at 43.95 µg/mL and above). All solvent (Minimum Essential Medium (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes and were considered acceptable for addition to the laboratory historical negative control data range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei with responses that are compatible with those in the laboratory historical positive control data range. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In the 4-hour and 24-hour exposure groups in the absence of S9, the test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, all values were within the 95% control limits of the historical control data, and no concentration-dependent trend was observed. In the 4-hour exposure group in the presence of S9, a statistically significant increase in the frequency of binucleate cells with micronuclei was observed at 468.75 µg/mL. However, all values were within the 95% control limits of the historical control data, and no concentration-dependent trend was observed when evaluated with a trend test. The response was therefore considered to be spurious and of no toxicological significance. The test item, Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA), was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro under the described test conditions. Please consider, that the provided results are still draft results. Currently, there are further discussions ongoing with respect to the significant increases in pH. An update will be provided to the authority as soon as the final study report will be available.

Gene mutation in mammalian cells:

A reliable study similar to OECD guideline 476 with the source substance TETA is available Slesinski, 1981). Preliminary experiments were performed to select an appropriate range of test concentrations in which the maximum concentration would allow survival of approximately 10% of the treated cells. A maximum concentration of 0.8% (by volume) was chosen for the highest dose-level and a total of seven concentrations of TETA were tested for mutation induction because a steep dose response was suggested from prescreening data. TETA produced a statistically significant increase in the frequency of mutations of CHO cells at several concentrations between 0.8% to 0.025% (by volume) in tests with and without the incorporation of a liver S9 metabolic activation system. The lack of a definite dose-related effect of treatment suggested that the alkaline effect of the test agent may have interfered with the tests. With S9 metabolic activation, the acidic S9 liver homogenate may have somewhat buffered the alkaline effect and a dose related trend in the mutation index was observed for treatments between 0.1% and 0.4%.

In conclusion, TEPA is considered to have a potential to induce gene mutation in mammalian cells.

DNA damage/repair:

One study similar to OECD 479 (in vitro sister chromatid exchange assay in mammalian cells) with Amines, polyethylenepoly-, tetraethylenepentamine fraction is available (Slesinski, 1987). The test substance produced a statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests with and without the incorporation of a metabolic activation system. An overall range of concentrations of 0.4 mg/mL to 3.0 mg/mL was used. The quantitative SCE increases were approximately the same in the tests with and without metabolic activation, and were 1.8 and 1.6 above control, respectively. Therefore, the TEPA is considered to produce a positive genotoxic effect in this test.

In vivo:

A reliable study similar to OECD guideline 474 with the source substance TETA is available (Guzzie, 1987). TETA was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female Swiss-Webster mice. Test doses for the micronucleus test were chosen from data obtained in a preliminary toxicity study with mice. Five doses of TETA ranging from 434 mg/kg bw to 900 mg/kg bw were administered as a single intraperitoneal (i .p.) injection. The LD50 dose was calculated from the cumulative mortality observed during a three day period after dosing. To select dose levels for the definitive micronucleus test, a combined LD50 value of approximately 740 mg/kg bw (651 to 877; 95% fiducial limits) was calculated by pooling the total number of deaths for males and females.

For the definitive micronucleus test, doses of 185 mg/kg bw, 370 mg/kg bw and 600 mg/kg bw were tested with both male and female Swiss-Webster mice. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that TETA did not produce positive or dose-related increases in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test. The absence of positive effects of TETA upon the incidence of micronuclei indicates that TETA does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.

Similar results were observed in a supporting study similar to OECD guideline 474 (SanSebastian, 1992). TETA was evaluated for potential clastogenic (chromosome-damaging) activity with the in vivo micronucleus test system employing both male and female CD-1 mice. In a preliminary test 50, 100, 250, 500 and 1000 mg/kg bw were tested via the intraperitoneal route. Pharmacotoxic signs were observed in the 100 mg/kg bw group. Mortality occurred in the 250 mg/kg bw group (1/4). All animals but one, died within 2 hours post-dose in the 500 and 1000 mg/kg bw dose groups. Therefore, 150 mg/kg bw was selected as test dose for the main study. Concurrent positive (triethylenemelamine) and negative (water) control agents, administered by i.p, injection, were used to demonstrate the reliability and sensitivity of the micronucleus test system. Results from the micronucleus determination demonstrated that TETA did not produce an increase in the incidence of micronuclei in peripheral blood polychromatic erythrocytes of the test animals at any of the sample periods tested. Data from the positive and negative control groups of animals demonstrated the appropriate responses for the animals in the test system consistent with a valid test. The absence of positive effects of TETA upon the incidence of micronuclei indicates that TETA does not possess clastogenic activity in vivo under the conditions of the micronucleus test system.

Further, an in vivo micronucleus test was carried out with TETA in mice using the intraperitoneal route (at levels of 130, 190 and 250 mg/kg bw) or the oral route (at levels of 1500, 3000 and 6000 mg/kg bw) which was described just briefly in published literature (Heinz, 1981). TETA was not mutagenic in the micronucleus test in vivo using both the oral and ip route.

Fifty chemicals were tested for mutagenic activity in post-meiotic and meiotic germ cells of male Drosophila melanogaster using the sexlinked recessive lethal (SLRL) assay among those also TETA (Foureman, 1994). As in the previous studies in this series, feeding was chosen as the first route of administration. If the compound failed to induce mutations by this route, injection exposure was used. Those chemicals that were mutagenic in the sex-linked recessive lethal assay were further tested for the ability to induce reciprocal translocations. Eleven of the 50 chemicals tested were mutagenic in the SLRL assay. TETA was ambiguous after feeding and negative after injection.

In conclusion, based on the above study results with the structural analogue substance TETA (CAS 90640-67-8) evidence is available to conclude that the registered substance Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7) is not mutagenic in vivo.

However, according to ECHA CCH-D-2114482145-49-01/F it cannot be finally concluded which material was indeed tested in the available in vivo studies, the source substance Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8) itself or the main constituent N,N'-bis(2-aminoethyl)ethane-1,2-diamine (CAS 112-24-3). Therefore, and to address the positive results obtained in the bacterial reverse mutation assay with Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7; key, Covance, 2021) a testing proposal for an in vivo genotoxicity study addressing gene mutation in somatic cells is included in the dossier. The final decision for the adequatein vivo follow-up test is not yet clear as currently, there are further investigations ongoing with respect to in vitro cytogenicity and also repeated dose toxicity. According to the CCH decision of ECHA (CCH-D-2114482142-55-01/F) a repeateddose oral toxicity study with the registered substance according to OECD 408 was requested by ECHA. This study is still in progress. Before a final conclusion on an adequate follow-up of a positive in vitro result can be drawn, not only the results from the in vitro testing should be reviewed, but also other relevant data on the substance (e.g. toxicokinetics, target organ specifity). Therefore, at least the in vitro micronucleus test should be finalised to see, if only mutagenicity should be addressed in vivo or also cytogenicity, also taking into consideration the 3Rs principle with respect to animal wellfare. To get information on (a) possible target tissue(s), the repeated dose oral toxicity study would be a good basis. Taken together, the final decison on the adequate in vivo follow-up gentotoxicity study is postponed until all relevant data necessary for the conclusion will be available.

Justification for classification or non-classification

Available data are not sufficient to draw a final conclusion on genetic toxicity of Amines, polyethylenepoly-, tetraethylenepentamine fraction (CAS 90640-66-7). Further data are necessary to conclude if classification according to Regulation (EC) No. 1272/2008 is required.