Amines, polyethylenepoly-, triethylenetetramine fraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Amines, polyethylenepoly-, triethylenetetramine fraction showed positive, ambiguous or negative results in several Ames tests, in vitro cytogenicity test (DRAFT), Sister Chromatide Exchange tests and UDS 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 - 31 August 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:

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)

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 : 1; OECD TG 471 permits non-repetition when a clear, positive response is obtained in the first mutation test (plate Incorporation method); therefore, a second, confirmatory test was not required;

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

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.

TREATMENT AND HARVEST SCHEDULE:
- 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:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

maximum increases over the vehicle control of 3.7 fold were noted in the absence of metabolic activation at 1500 µg/plate and 4.4 fold in the presence of metabolic activation at 5000 µg/plate

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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

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

Remarks:

maximum increases over the vehicle control of 18.8 and 13 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate

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:

positive

Remarks:

maximum increases over the vehicle control of 3.4 and 1.9 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate

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:

negative

Remarks:

minor statistically significant increases were noted in the absence (max = 1.9 fold) and presence (max= 2.5 fold) of metabolic activation; increases did not meet the required criteria (> 3-fold increase), but were considered in the overall assessment;

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:

negative

Remarks:

minor statistically significant increases were noted in the absence of metabolic activation (max = 2.5 fold); increase did not meet the required criteria (> 3-fold increase), but were considered in the overall assessment;

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).
- 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. The revertant colony counts for TA1537 dosed with 9-Aminoacridine in the absence of metabolic activation (Experiment 1) were above the historical values. This is considered not to affect study integrity as the strain responded within acceptable parameters for the vehicle and untreated controls and viability data. 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).
- Results: Dose-related and statistically significant increases in excess of 2-fold were noted in bacterial strains TA100 and WP2uvrA in both the absence and presence of metabolic activation. For TA100, maximum increases over the vehicle control of 3.7 fold were noted in the absence of metabolic activation at 1500 µg/plate and 4.4 fold in the presence of metabolic activation at 5000 µg/plate. For WP2uvrA, maximum increases over the vehicle control of 18.8 and 13 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. For TA98, maximum increases over the vehicle control of 3.4 and 1.9 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. All of these increases were also accompanied by individual revertant colony counts above the upper maxima of the historical vehicle and untreated control ranges of the lab. Other, minor statistically significant increases were noted for TA1535 in the absence (max = 1.9 fold) and presence (max= 2.5 fold) of metabolic activation and TA1537 in the absence of metabolic activation (max = 2.5 fold). Whilst these increases did not all meet the required criteria (3-fold for TA1535 and TA1537, and 2-fold for the remaining strains), there were signs of a
mild response, which should also be considered in the overall assessment of the results.
See Tables 1 and 2 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)	113 145 121	(126) 16.7#	20 19 20	(20) 0.6	27 26 28	(27) 1.0	21 27 21	(23) 3.5	10 12 8	(10) 2.0
1.5 µg	126 136 138	(133) 6.4	24 28 15	(22) 6.7	18 21 22	(20) 2.1	32 26 33	(30) 3.8	8 12 15	(12) 3.5
5 µg	143 122 134	(133) 10.5	25 19 20	(21) 3.2	21 25 20	(22) 2.6	22 32 24	(26) 5.3	11 15 8	(11) 3.5
15 µg	146 132 141	(140) 7.1	22 19 20	(20) 1.5	41 31 30	(34) 6.1	30 39 25	(31) 7.1	17 9 11	(12) 4.2
50 µg	207 208 233	(216) 14.7 ***	20 26 15	(20) 5.5	116 82 110	(103) 18.1 ***	29 40 39	(36) 6.1	9 15 10	(11) 3.2
150 µg	315 285 310	(303) 16.1 ***	28 20 34	(27) 7.0	168 159 183	(170) 12.1 ***	44 27 42	(38) 9.3	7 7 12	(9) 2.9
500 µg	378 401 416	(398) 19.1 ***	41 29 31	(34) 6.4	295 325 307	(309) 15.1 ***	39 64 62	(55) 13.9 ***	9 17 9	(12) 4.6
1500 µg	465 467 455	(462) 6.4 ***	31 32 48	(37) 9.5 *	379 391 364	(378) 13.5 ***	46 51 70	(56) 12.7 ***	13 10 14	(12) 2.1
5000 µg	468 435 454	(452) 16.6 ***	22 40 38	(33) 9.9 *	473 527 523	(508) 30.1 ***	63 81 92	(79) 14.6 ***	17 24 18	(20) 3.8 *
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	649 696 624	(656) 36.6	267 269 203	(246) 37.5	152 152 148	(151) 2.3	112 114 137	(121) 13.9	1880 1495 1605	(1660) 198.3

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)	127 131 125	(128) 3.1#	26 16 9	(17) 8.5	17 33 29	(26) 8.3	37 41 37	(38) 2.3	15 9 18	(14) 4.6
1.5 µg	144 127 118	(130) 13.2	11 12 24	(16) 7.2	31 25 22	(26) 4.6	34 43 30	(36) 6.7	7 11 10	(9) 2.1
5 µg	146 142 133	(140) 6.7	15 15 8	(13) 4.0	19 18 27	(21) 4.9	29 25 32	(29) 3.5	19 11 13	(14) 4.2
15 µg	157 176 168	(167) 9.5 **	19 17 26	(21) 4.7	27 25 20	(24) 3.6	46 47 46	(46) 0.6	18 8 16	(14) 5.3
50 µg	234 201 218	(218) 16.5 ***	19 19 13	(17) 3.5	67 61 79	(69) 9.2 ***	37 31 26	(31) 5.5	15 11 12	(13) 2.1
150 µg	356 384 384	(374) 15.4 ***	35 22 17	(25) 9.3	148 124 123	(132) 14.2 ***	50 39 36	(42) 7.4	19 11 8	(13) 5.7
500 µg	521 462 471	(485) 31.8 ***	21 25 24	(23) 2.1	202 209 214	(208) 6.0 ***	44 57 50	(50) 6.5 *	10 13 11	(11) 1.5
1500 µg	503 507 489	(500) 9.5 ***	22 25 41	(29) 10.2	269 287 276	(277) 9.1 ***	65 68 53	(62) 7.9 ***	18 13 12	(14) 3.2
5000 µg	546 539 606	(564) 36.8 ***	32 43 30	(35) 7.0 *	357 303 355	(338) 30.6 ***	70 73 77	(73) 3.5 ***	15 11 15	(14) 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	1531 1677 1733	(1647) 104.3	316 315 268	(300) 27.4	152 174 196	(174) 22.0	116 129 104	(116) 12.5	247 263 269	(260) 11.4

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

** p≤0.01

*** p≤0.001

# Standard deviation

Conclusions:

positive with and without metabolic activation

Amines, polyethylenepoly-, triethylenetetramine fraction (TETA) was positive in the Ames/Salmonella Plate Incorporation 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-, triethylenetetramine fraction (TETA) induced dose-related and statistically significant increases in the frequency of TA100, TA98 and WP2uvrA revertant colonies that met the criteria for a positive result, both with and without metabolic activation (S9-mix). Minor statistically significant increases were noted for TA1535 in the absence and presence of metabolic activation and TA1537 in the absence of metabolic activation. These increases did not meet the required criteria for a positive outcome. They were regarded as signs of a mild response and as such taken into account for the overall assessment. Under the conditions of this test Amines, polyethylenepoly-, triethylenetetramine fraction (TETA) was therefore 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:

25 August 2020 - 22 October 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 24 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*, 78.13, 156.25, 312.5*, 625*, 937.5*, 1250* µg/mL
- 4 h / +S9: 0*, 78.13, 156.25, 312.5*, 625*, 937.5*, 1250* µg/mL
- 24 h / -S9: 0*, 78.13, 156.25, 312.5*, 625*, 937.5*, 1250* 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 1250 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. The test item formulations were dosed to give a final concentration of 10% in the cultures. A correction for the purity of the test item of 97.6% 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); an additional 1000 binucleate cells were scored for each replicate of the 24-hour exposure group in the absence of S9 from the main experiment due to inconsistencies in responses between the ‘A’ and ‘B’ cultures and to add additional statistical power to the data and clarify any response;
- 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:

no cytotoxicity

Remarks:

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 1250 µg/mL due to pH (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, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 937.5, and 1250 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 1250 µg/mL in all three of the exposure groups. The test item induced evidence of modest toxicity in the 24-hour exposure group only (30% cytostasis at 1250 µg/mL). 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. 1250 µg/mL, due to marked increases in pH, in all three of the exposure groups.

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. All of the acceptability criteria were met. 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 modest 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 marked 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, very modest dose-related toxicity was observed with 25%, 32% and 29% cytostasis at 625, 937.5 and 1250 µg/mL, respectively. The maximum dose level selected for scoring of micronuclei in the binucleate cells was 1250 µg/mL for all three exposure groups. However, due to causing a pH change of more than 1 pH unit the 1250 µg/mL dose level was subsequently excluded from the analysis.

- Genotoxicity results: In the 4-hour exposure groups, in both the absence and presence of S9, the test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, up to 937.5 µg/mL. All values were within the 95% control limits of the historical control data, and no concentration-dependent trend was observed. The dose level of 1250 µg/mL was excluded from the final analysis due to a pH change of greater than 1 unit which was considered to invalidate the results at this dose level.
In the 24-hour exposure group in the absence of S9, an additional 1000 binucleate cells were scored for each of the replicate cultures due to consistencies in response between the replicates and to add statistical power to the data. A small but statistically significant increase in the frequency of binucleate cells with micronuclei was observed at 937.5 µg/mL, which was due mainly to increases in the ‘B’ culture only. The increase observed marginally exceeded the upper 95% control limit of the historical control data for the 24-hour exposure group, but was within the upper limit of the laboratory’s historical control range and the value observed would be considered acceptable for inclusion in the historical solvent control data for the 24-hour exposure group. There was also no concentration-dependent trend observed when evaluated with a trend test. The response was therefore considered to be spurious and of no toxicological significance. The data generated from the dose level of 1250 µg/mL was again excluded from the final analysis due to the excessive pH change of more than 1 unit which meant that the results could not be relied on.
Limiting the upper dose concentrations tested based on a marked pH change is considered to be consistent with the requirements of the OECD 487 guideline, which states, that concentrations which have the capability of producing artefactual positive results such as marked changes in pH should be avoided. Therefore, it is considered that 937.5 µg/mL is the maximum practical dose level that could be analyzed in this study to provide reliable results.

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	39.06	19.53	78.13	156.25	625	937.5	1250	2500	5000
pH	7.39	7.38	7.26	7.44	7.41	7.94	8.10	8.59	9.42	9.91
Osmolality	315	315	313	317	315	310	319	303	318	336

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	1.80 1.85 1.77 1.78	1.80	0	0.30 0.20 0.70 0.60	0.45	/	0.612
	312.5	A B	1.81 1.78	1.80	1	0 0.60	0.30	/
	625	A B	1.65 1.79	1.72	10	0.50 0.40	0.45	/
	937.5	A B	1.82 1.76	1.79	1	0.40 0.80	0.60	0.44
	1250^d	A B	1.67 1.68	1.68	16	0.10 0.90	0.50	/	/
	MMC 0.2	A B	1.66 1.68	1.67	16	6.10 4.0	5.05	2.01E-33***	/
4-hour +S9	Vehicle (MEM)	A1 A2 B1 B2	1.84 1.90 1.77 1.85	1.84	0	0.30 0.30 0.10 0.10	0.20	/	0.147
	312.5	A B	1.67 1.82	1.75	11	0.20 0.10	0.15	/
	625	A B	1.74 1.71	1.73	14	0.20 0.70	0.45	0.0859
	937.5	A B	1.61 1.69	1.65	23	0.20 0.60	0.40	-
	1250^d	A B	1.82 1.67	1.75	11	0.60 0.50	0.55	0.0229*	-
	CP 5	A B	1.43 1.55	1.49	42	2.20 4.90	3.55	7.25E-27***	-
24-hour -S9	Vehicle (MEM)†	A1 A2 B1 B2	1.65 1.72 1.80 1.89	1.77	0	0.15 0.45 0.15 0.50	0.31	-	0.072
	312.5†	A B	1.69 1.63	1.66	14	0.30 0.05	0.18	-
	625†	A B	1.62 1.52	1.57	25	0.60 0.60	0.60	0.0758
	937.5†	A B	1.53 1.51	1.52	32	0.45 1.00	0.73	0.0015**
	1250^d	A B	1.51 1.57	1.54	29	0.80 1.30	1.05	1.95E-05***	-
	DC 0.075†	A B	1.30 1.35	1.33	58	5.50 4.80	5.15	7.338E-74***	-

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

^dThe dose level of 1250 µg/mL was excluded from the final analysis due to a pH change of greater than 1 unit which was considered to invalidate the results at this dose level.

* P<0.05

** P<0.01

*** P<0.001

† Additional 1000 binucleate cells scored per replicate culture

Conclusions:

negative - DRAFT results

Executive summary:

The test item, Amines, polyethylenepoly-, triethylenetetramine fraction (TETA), 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, using a dose-range which was limited to the maximum practical dose level (highest dose evaluated was 937.5 µg/mL). The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro under the described test conditions (DRAFT results). To further evaluate the effect of pH shift on the outcome of the test, the study will be repeated with adjusting the pH. Due to pH adjustment also higher test substance concentrations can be included. The results of this study ammendment will be included in the draft study report. Thus, the provided results are preliminary results and an update will be provided to the authority as soon as the final report will be available.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In three in vivo Micronucleus Tests no genotoxic potential of Amines, polyethylenepoly-, triethylenetetramine fraction were seen. In a Drosophila SLRL test Amines, polyethylenepoly-, triethylenetetramine fraction showed ambiguous results after feeding exposure and negative results after test substance injection.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 10, 1987 - April 17, 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

No information of substance purity or composition. In appendix I of the report analytical information has been added but that is an MS printout, purity 68.5%? The study followed GLP and was performed according to methods similar to OECD 474. 1000 instead of 2000 immature erythocytes were examined per animal. No bone marrow toxicity was observed; however, at higher dose levels as administered in this test mortality occurred.

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency - Health Effect Test Guidelines, EPA Report 560/6-83-001

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

- 1000 instead of 2000 immature erythocytes were examined per animal.

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles Rivers Laboratories, Portage, MI
- Age at study initiation: 5 weeks
- Weight at study initiation: male 23.6 g to 26.6 g, female 20.5 g to 23.5 g.
- Assigned to test groups randomly: yes, under following basis: randomized by weight and animals outside a range of two standard deviations from the mean were not used.
- Fasting period before study: not applicable
- Housing: Five mice/sex/cage were housed in shoe-box type plastic cages, measuring 30 x 20 x 12.5 cm.
- Diet (e.g. ad libitum): ad libitum with a basic diet of Agway PROLAB@ Animal Diet
- Water (e.g. ad libitum): Municipal Authority of Westmoreland County (Greensburg, PA) and was available ad libitum.
- Acclimation period: 5-6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): - Humidity (%): In the opinion of the study director, there were no unacceptable variations in temperature or humidity during the testing periods which would adversely affect the quality or integrity of the study.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: March 10, 1987 - April 17 1987

Route of administration:

intraperitoneal

Vehicle:

water

Details on exposure:

None

Duration of treatment / exposure:

na

Frequency of treatment:

single i.p. injection.

Post exposure period:

72 hours

Dose / conc.:

185 mg/kg bw/day (nominal)

Dose / conc.:

370 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

triethylenemelamine
- Justification for choice of positive control(s): standard positive control
- Route of administration: i.p.
- Doses / concentrations: 0.3 mg/kg bw

Tissues and cell types examined:

Blood from the tail fo the mice

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: range finding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Three dose levels of approximately 80%, 50% and 25% of the pooled LD50 value were evaluated for effects upon the incidence of micronuclei. A single i.p. injection was given. Blood samples were taken at 3 time periods at approximately 30, 48 and 72 hr after dosing.

DETAILS OF SLIDE PREPARATION: One or two blood smear slides were prepared for each animal sampling time. Micronuclei in peripheral blood, polychromatic erythrocytes were stained with Gurr's R-66 Giemsa diluted in phosphate buffer. Slides were coded by animal number only and read blindly to prevent bias.

METHOD OF ANALYSIS:
A minimum of 1000 polychromatic erythocytes was examined microscopically for each animal per sample time, unless cytotoxicity of the test material prevented this goal. The polychromatic:normochromatic erythrocyte ratio for approximately 1000 total cells was calculated and recorded and these data are summarized in the final report as an estimate, of cytotoxicity of the test agent.
Micronuclei were identified as darkly-stained, spherical, inclusions in polychromatic erythrocytes. Polychromatic, erythrocytes were identified by the pale-bluish staining of the cytoplasm in contrast to the lack of blue stain for normochromatic erythrocytes.

Statistics:

Data were compared for significant differences from the vehicle control frequencies using the Fisher's Exact Test (Sokal and Rohlf, 1981). Data for
males and female mice at each sample perfod were combined for statistical analyses because Analysis of Variance tests showed that there was.no
significant difference in micronuclei frequencies between sexes at each sample period. A positive result in the micronucleus test was concluded if at least one statistically significant (p ≤0.01) increase above the vehicle control was .observed with an indication of a dose-related effect of treatment. A test was considered to be inconclusive if only one dose produced effects statistically different from the control (0.05 ≥ p ≥ 0.01) and a dose-effect relationship was apparent. A test result was considered to be negative if no statistically significant differences were apparent between the vehicle control and groups of animals treated with TETA.

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

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Analysis of variance testing indicated that there was no significant difference between the mortality response of male and female mice treated with TETA, Thus, a combined LD50 value of approximately 740 mg/kg was calculated and used to determine the test doses for the definitive micronucleus test. The lower and upper 95% fiducial limits for the LD50 value were 651 mg/kg and 877 mg/kg, respectively.
The PCE/NCE ratio of the vehicle control and the highest test dose with adequate numbers of survivors (≥ 3) was quantified and compared to determine possible bone marrow cytotoxicity. At 48 hrs after dosing; the PCE/NCE ratios of both the male and female mice were similar to the vehicle control values, thus indicating the absence of bone marrow toxicity. Since no bone marrow toxicity was evident at this sample period, no additional blood smears were obtained at later time intervals

RESULTS OF DEFINITIVE STUDY
Micronucleus determinations were conducted using a minimum of 5 animals/sex/group. Additional animals were added to some groups because deaths were expected at the higher dosages. However, extra animals were assessed for micronucleus frequencies only as needed to assure that a total of five animals are evaluated. Three dose levels of 600 mg/kg, 370 mg/kg and 185 mg/kg were selected for testing in the definitive micronucleus test at approximately 80%, 50% and 25% of the combined male-female LD50 value, respectively.

No remarkable decreases in the PCE/NCE ratios relative, to the control values were observed in this study at any of the three sampling periods. PCE/NCE ratios of the male animals treated with TEM were lower than the concurrent negative control values which is an expected and typical finding because of the cytotoxicity and clastogenicity of this agent.

Analysis of variance (AOV) testing indicated that the data for male and female mice sampled at 30 hr, 48 hr or 72 hr after dosing were not statistically different; thus, values were pooled for Fisher's Exact analyses. No statistically significant or treatment-related increases in the numbers of micronuclei were observed with any of the treatment groups sampled at any of the sample intervals following injection of the test chemical.

TEM, used as a positive control agent for this study, produced highly significant increases in numbers of. micronuclei demonstrating the appropriate sensitivity of the test system. Numbers of micronuclei in the vehicle control animals were in a low and acceptable range for this test system at all sampling times.

Conclusions:

Interpretation of results: negative
Test results for this study showed that TETA was not an active agent in producing treatment-related increases in micronuclei in male and female Swiss-Webster mice. Relatively high dosage levels of TETA were evaluated no treatment-related clastogenic activity was observed. TETA was considered to be inactive as a clastogenic agent in vivo under the conditions of the micronucleus test.

Executive summary:

Triethylenetetramine (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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

Gene mutation in bacteria:

Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8) 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). Dose-related and statistically significant increases in excess of 2-fold were noted in bacterial strains S. typhimurium strains TA100 and E. coli WP2uvrA in both the absence and presence of metabolic activation (S9-mix) using the plate incorporation method. For S. typhimurium strain TA100, maximum increases over the vehicle control of 3.7 fold were noted in the absence of metabolic activation at 1500 µg/plate and 4.4 fold in the presence of metabolic activation at 5000 µg/plate. For E. coli WP2uvrA, maximum increases over the vehicle control of 18.8 and 13 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. For S. typhimurium strain TA98, maximum increases over the vehicle control of 3.4 and 1.9 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. All of these increases were also accompanied by individual revertant colony counts above the upper maxima of the historical vehicle and untreated control ranges. Other, minor statistically significant increases were noted for S. typhimurium strain TA1535 in the absence (max = 1.9 fold) and presence (max= 2.5 fold) of metabolic activation and for S. typhimurium strain TA1537 in the absence of metabolic activation (max = 2.5 fold). These increases did not meet the required criteria (3-fold for TA1535 and TA1537). Therefore, they were regarded as mild response and were considered as such in the overall assessment of the results. 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-, triethylenetetramine fraction (TETA) induced dose-related and statistically significant increases in the frequency of S. typhimurium TA100, TA98 and E. coli WP2uvrA revertant colonies that met the criteria for a positive result, both with and without metabolic activation. A second mutation test using the preincubation method was not performed, because OECD guideline 471 permits non-repetition when a clear, positive response is obtained in the first mutation test. Under the conditions of this test, Amines, polyethylenepoly-, triethylenetetramine fraction (TETA) was therefore considered to be mutagenic.

There are several additional studies, which support the positive outcome of Amines, polyethylenepoly-, triethylenetetramine fraction in the bacterial reverse mutation assay (Stankowski, 1992, Guzzie, 1987 and Willems, 1980; all conducted similar to OECD guideline 471) in different S. typhimurium strains with and without metabolic activation.

Available reports of Amines, polyethylenepoly-, triethylenetetramine fraction in bacterial reverse mutation assays with limited documentation or limitations in the study design were disregarded due to major methodological deficiencies.

In vitro cytogenicity:

Amines, polyethylenepoly-, triethylenetetramine fraction (CAS 90640-67-8) 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 maximum concentration selected for the 4-hour exposure groups in the absence and presence of S9 and the 24-hour exposure group was limited to 1250 µg/mL due to significant increases in pH of more than 1 pH unit at 1250 µg/mL and above. The dose levels selected for the Main Experiment were as follows: 0*, 78.13, 156.25, 312.5*, 625*, 937.5* and 1250* µg/mL (*Dose levels selected for evaluation of micronucleus frequency in binucleate cells). 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. In the 24-hour exposure group in the absence of S9, dose-related toxicity was observed with 25%, 32% and 29% cytostasis at 625, 937.5 and 1250 µg/mL. All solvent (Minimal Essential Medium) 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 exposure groups, in both the absence and presence of S9, the test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei up to the dose level of 937.5 µg/mL. All values were within the 95% control limits of the historical control data, and no concentration dependent trend was observed. In the 24-hour exposure group in the absence of S9, a statistically significant increase in the frequency of binucleate cells with micronuclei was observed at 937.5 µg/mL. The increase observed marginally exceeded the upper 95% control limit of the historical control data for the 24-hour exposure group. However, the value observed was within the laboratory’s maximum historical control limit for a solvent control in the 24-hour exposure group and the value observed would be considered acceptable for inclusion in the historical solvent control data for the 24-hour exposure group. There was also no concentration-dependent trend observed when evaluated with a trend test and the response at 937.5 µg/mL was due to an increase mainly in one replicate. The response was therefore considered to be spurious and of no toxicological significance. The dose level of 1250 µg/mL was scored in all three exposure groups, but has been excluded from any analysis as it is considered that any data generated would not be reliable or valid due to a pH change of greater than 1 pH unit which may have an impact on the result. The OECD 487 Guideline states that concentrations that have the capability of producing artefactual results such as a marked change in pH should be avoided. However, to further evaluate the effect of pH shift on the outcome of the test, the study will be repeated with adjusting the pH. Due to pH adjustment also higher test substance concentrations can be included. The results of this study ammendment will be included in the draft study report. Thus, the provided results are preliminary results and an update will be provided to the authority as soon as the final report will be available.

Gene mutation in mammalian cells:

A reliable study similar to OECD guideline 476 with the test substance Amines, polyethylenepoly-, triethylenetetramine fraction 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 a proximately 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 the test substance were tested for mutation induction because a steep dose response was suggested from prescreening data. The test substance 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%.

DNA damage/repair:

Three reliable studies are available similar to OECD 479 (in vitro sister chromatid exchange assay in mammalian cells (Slesinski, 1981 and 1987). Amines, polyethylenepoly-, triethylenetetramine fraction produced a highly statistically significant and dose-related increase in the frequency of SCE in CHO cells in tests without the incorporation of a metabolic activation system. With metabolic activation, the frequency of SCE was decreased, was found to be lower than without metabolic activation, but the highest dose level produced a highly statistically significant effect. An overall range of concentrations of 0.4% to 0.0125% (by volume) was used. The high frequency of SCE observed in the test without metabolic activation indicates that the test substance is mutagenic in CHO cells (Slesinski, 1981). TETA - Sample A (Slesinski, 1987), which is considered to be Amines, polyethylenepoly-, triethylenetetramine fraction, produced dose-related and statistically significant increases in SCEs in the test without addition of a rat-liver S9 metabolic activation system. With S9 activation, an inverse dose response relationship was observed and a significant response was obtained only at the lowest test concentration. The highest increases in SCEs above the combined solvent control values were approximately 1.4 fold without S9 and 1.3 fold with S9 activation. No remarkable degree of cell-cycle inhibition was produced by the test chemical by determination of the ratio of numbers of cells in the first and second cycle of division. The test chemical was considered to be a positive but weakly-active genotoxic agent in the SCE test system. Triethylenetetramine Raney Nickel Treated (TETA-RNT), which is also considered to belong to the substance identity of Amines, polyethylenepoly-, triethylenetetramine fraction, produced a statistically significant and dose-related effect upon the frequency of SCE in CHO cells in tests both with and without the incorporation of an S9 metabolic activation system. An overall range of concentrations between 0.025% to 0.5% (by volume) was tested and the effects on the SCE frequency were determined with the highest five concentrations which allowed adequate cell division. The results indicated that TETA-RNT was an active agent in this test and should be considered a probable positive mutagenic agent for production of DNA damage in animal cells in culture (Slesinski, 1981).

In conclusion, Amines, polyethylenepoly-, triethylenetetramine fraction is considered to have a potential to induce DNA damage in mammalian cells.

Four reliable studies are available similar to OECD 482 (unscheduled DNA synthesis in mammalian cells, Slesinski, 1981, Schumann, 1979, Pharmakon, 1992). Amines, polyethylenepoly-, triethylenetetramine fraction produced statistically significant increases in the amount of UDS activity in evaluations of concentrations between 1% and 0.001% (by volume). The test substance was considered to be active in the test with the hepatocyte test system and positive effects were observed in tests using both nuclei and DNA to detect increases in UDS (Slesinski, 1981). TETA-RNT produced slight increases in the amount of UDS activity in evaluations of concentrations between 1% and 0.001% (by volume). TETA-RNT was considered to be weakly active in the test with the hepatocyte test system because a majority of the UDS levels were significantly greater than historical negative control values for this test system (Slesinski, 1981). The genotoxic potential of product grade triethylenetetramine (TETA) and distilled-TETA was evaluated in the rat hepatocyte unscheduled DNA synthesis (UDS) assay (Schumann, 1979). Neither TETA nor distilled-TETA elicited significant UDS at concentrations of 0.1, 0.01, 0.001, 0.00001, 10E-5, 10E-6, 10E-7 or 10E-8 M. Alterations in the hepatocyte cultures indicative of toxicity were observed with both TETA and distilled-TETA at a concentration of 0.1 M. The appearance of the cultures improved with decreasing concentrations of TETA and distilled-TETA so that cultures exposed to 10E-3 to 10E-8 M appeared comparable to control cultures. The inability of TETA or distilled-TETA to elicit DNA repair over the wide spectrum of concentrations tested indicates a lack of genotoxicity under the conditions of the assay. A further UDS test was carried out with Amines, polyethylenepoly-, triethylenetetramine fraction in rat hepatocytes (Pharmakon, 1992). Analysis of the data for the test substance did not produce mean net nuclear grain counts >= 5 at any of the doses scored. In addition, the percentage of hepatocytes in repair ranged from 4 -8%. The negative and positive control values were 13.5 +/- 11.1 and 22.8 +/- 12.8 with 4 and 92.7% hepatocytes in repair, respectively. These values were within the criteria for a valid test. Under the conditions of this assay, the test substance did not induce unscheduled DNA synthesis (repair) in rat primary hepatocytes at concentrations up to 200 µg/mL.

In conclusion, Amines, polyethylenepoly-, triethylenetetramine fraction is considered to have a potential to induce unscheduled DNA synthesis (repair) in mammalian cells.

In vivo:

A reliable study similar to OECD guideline 474 with Amines, polyethylenepoly-, triethylenetetramine fraction is available (Guzzie, 1987). The test substance 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 the test substance 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 the test substance 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 the test substance upon the incidence of micronuclei indicates that the test substance 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). Amines, polyethylenepoly-, triethylenetetramine fraction 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 the test substance 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 the test substance upon the incidence of micronuclei indicates that the test substance 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 Amines, polyethylenepoly-, triethylenetetramine fraction 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 Amines, polyethylenepoly-, triethylenetetramine fraction (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. The test substance was ambiguous after feeding and negative after injection.

 

Based on the above study results evidence is available to conclude that the Amines, polyethylenepoly-, triethylenetetramine fraction 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 registered 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-, triethylenetetramine fraction (CAS 90640-67-8; 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 adequate in vivo follow-up test is not yet clear as currently, there are further investigations ongoing with respect to in vitro cytogenicity (please see also above, section ‚In vitro cytogenicity‘ for further details) and also repeated dose toxicity. According to the CCH decision of ECHA (CCH-D-2114482145-49-01/F) a repeated dose 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-, triethylenetetramine fraction (CAS 90640-67-8). Further in vivo data are necessary to conclude if classification according to Regulation (EC) No. 1272/2008 is required.