Reaction product of 1,3,5-Triazine-2,4,6-triamine, polymer with formaldehyde, methylated and C16-18 fatty alcohols

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb - 04 Apr 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

EC Number:

947-918-6

Molecular formula:

not applicable, UVCB substance.

IUPAC Name:

Reaction product of C16-18 (even numbered) alcohols with reaction products of 1,3,5-Triazine, 2,4,6,-triamine, polymer with formaldehyde, methylated

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Remarks:

not applicable

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, induced with phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

The test item was tested in the pre-experiment with tester strains TA 98 and TA 100 with the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
No cytotoxicity was observed.

Experiment I - Plate Incorporation Method (all tester strains): 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed.

Experiment II - Plate Incorporation Method (all tester strains): 15.8, 50.0, 158, 500, 1580 and 5000 µg/plate
The maximum concentration was chosen as recommended in the guideline followed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF)
- Lot/Batch: 17A261139

- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

A. dest.

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD): -S9: 10 µg/plate (in DMSO) for TA 98 and 40 µg/plate (in DMSO) for TA 1537; 2-aminoanthracene (2-AA): +S9: 2.5 µg/plate (in DMSO) for TA 98, TA 100, TA 1535, TA 1537 and 10 µg/plate (in DMSO) for TA 102

Remarks:

Biological activity of the S9 mix in the Salmonella typhimurium assay was assessed using 2-aminoanthracene and benzo[a]pyrene.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation for experiments I and II)

DURATION
- Exposure duration: at least 48 h (at 37 °C)

NUMBER OF REPLICATIONS:
triplicate in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of clearing or diminution of the background lawn or of reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:

VALIDITY CRITERIA:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the negative control plates (A. dest.) +/-S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

EVALUATION CRITERIA:
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

The colonies were counted using a ProtoCOL counter (Meintrup DWS Laborgeräte GmbH). If precipitation of the test item precluded automatic counting the revertant colonies were counted by hand. In addition, tester strains with a low spontaneous mutation frequency like TA 1535 and TA 1537 were counted manually. Mean values and standard deviations of each treatment and control (negative, positive and solvent) were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

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

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

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

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

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

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment at any concentration

HISTORICAL CONTROL DATA: Results of positive and negative controls fell within historical control data range. Data are summarised in "Any other information on results incl. tables".

Any other information on results incl. tables

Due to the amount and complexity of the results tables, all additional data are attached in a pdf document (Tabulated results.pdf). Only the historical laboratory control data are summarised below.

Table 1: Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) without S9 (-S9)

 

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	24.2	90.7	13.8	8.2	270.4
SD	6.7	15.6	6.7	2.9	55.0
Min	11	49	4	3	141
Max	58	155	41	35	472
RSD [%]	27.7	17.2	48.6	35.3	20.3
n	972	1191	929	931	682

Table 2: Historical Laboratory Control Data of the Positive Control (in 2014

- 2016) without S9 (-S9)

 

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Substance Conc./plate	4-NOPD 10 µg	NaN3 10 µg	NaN3 10 µg	4-NOPD 40 µg	MMS 1 µL
Mean	430.7	612.1	792.0	94.5	1729.2
SD	155.5	220.0	299.5	22.7	518.8
Min	141	132	38	35	272
Max	1830	1423	1854	273	3321
RSD [%]	36.1	35.9	37.8	24.0	30.0
n	971	1188	931	929	682

 

Table 3: Historical Laboratory Control Data of the Negative Control (in 2014 - 2016) with S9 (+S9)

 

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Mean	29.0	96.4	10.5	8.3	339.7
SD	6.8	14.1	4.5	3.1	71.3
Min	15	62	3	3	157
Max	59	160	38	36	586
RSD [%]	23.4	14.6	42.7	37.4	21.0
n	967	1189	925	926	676

 

Table 4: Historical Laboratory Control Data of the Positive Control (in 2014

- 2016) with S9 (+S9)

 

	TA 98	TA 100	TA 1535	TA 1537	TA 102
Substance Conc./plate	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 2.5 µg	2-AA 10 µg
Mean	1880.5	1727.7	133.9	234.1	801.2
SD	708.5	522.0	134.9	101.4	223.7
Min	70	169	22	26	137
Max	3606	3132	1954	682	3588
RSD [%]	37.7	30.2	100.8	43.3	27.9
n	966	1184	927	925	678

 

S9: metabolic activation

Mean: mean of revertants/plate

Min.: minimum of revertants/plate

Max.: maximum of revertants/plate

SD: Standard Deviation

RSD: Relative Standard Deviation

n: Number of control values

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames Assay the substance was not mutagenic in any of the five bacterial strains (TA1535, TA1537, TA98, TA100 and TA102) with and without metabolic activation tested.