6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:3)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The acid component CAS 80584-91-4 did not increase the mutagenic frequency in an Ames test performed in Salmonella strains TA 100, T A98, TA 1535, TA 1537, TA 1538 and in Escherichia coli WP2uvrA with up to 5000 μg test substance per plate in experiments with and without microsomal activation. The test substance did not induce mutation at the HPRT locus of V79 Chinese Hamster lung cells in the presence or absence of S9 mix with concentrations of up to 480 µg/ml. No induction of chromosome aberrations at non-cytotoxic concentrations was observed in a chromosome aberration assay according to OECD TG 473 in Chinese hamster ovary cells. In the presence of S9 mix at precipitating concentrations that were deemed toxic to the cells, a positive response was reported.

TEA did not cause gene mutations in Salmonella typhimurium and Escherichia coli (Ames test), nor were chromosomal aberrations or sister chromatid exchanges induced in Chinese hamster ovary cells. An in vitro gene mutation assay (mouse lymphoma (L5178Y TK+/-) forward gene mutation assay) was also negative. All tests were performed in the absence and presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 24,2012 - August 14,2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Remarks:

hameln rds a.s., Section of Biological Studies, Hormi 36, 900 01 Modra, Slovak Republic, Department of Microbiology

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbecco's Modified Eagle's medium (DMEM) with 4.5 g/L of glucose supplemented with L-glutamine, Penicillin, Streptomycin and 10 % FBS (growth medium only)
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (S-9) prepared from 20-methylcholanthrene induced Sprague-Dawley rats.

Test concentrations with justification for top dose:

Range finder: 0; 0.75; 7.5; 15; 30; 60; 120; 240; 480 µg/ml
Main experiment I: 0; 3.75; 7.5; 15; 30; 60; 120 µg/ml (-S9); 0; 7.5; 15; 30; 60; 120; 240; 480 µg/ml (+S9)
Main experiment II: 0; 7.5; 15; 30; 60; 120; 240; 480 µg/ml (+S9 and -S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: solubility considerations

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

with and without S9

True negative controls:

no

Positive controls:

yes

Remarks:

with and without S9

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

DMBA: 3 µg/ml with S9; EMS: 600 µg/ml without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours with and without S9-mix
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 10 µg/mL of 6TG

NUMBER OF REPLICATIONS: single cultures

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in Chinese hamster lung V79 cells if:
1. The mutant frequency at one or more concentrations was at least 3-fold greater than that of the negative control
2. Concentration-related increase in mutant frequency
3. The effects described above were reproducible.

Statistics:

Multiple sample comparison of treated and untreated cell sets was processed applying Kruskal-Wallis test. The P-value from the test was considered to draw the relevant conclusion about statistical significance. Significance level of p<0.01 was taken into account. Multiple sample comparison was followed by two sample test applying Mann-Whitney W test to compare the medians of the two samples. The difference between medians was considered statistically significant at p<0.01. All individual values of frequencies are presented together with summary statistics involving count, average, standard deviation, coefficient of variation, minimum, maximum, range and standard skewness.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes of pH of treatment media were observed in the Range-Finder experiments at concentrations up to 480 µg/mL tested as compared to the concurrent vehicle controls.
- Effects of osmolality: The osmolality values were within the physiological ranges for these cells.
- Precipitation: No precipitate was observed in any experiment upon addition of the test article to the cultures or at the end of the 3 hour incubation period.

RANGE-FINDING/SCREENING STUDIES:
In the initial cytotoxicity Range-Finder experiment up to eight concentrations were tested in the absence of S-9 ranging from 0.75 to 480 µg/mL. At these concentrations, reductions in RPE values (%RPE reduced to 10 to 20%) were not achieved. Based on these results, the concentrations ranging from 3.75 µg/mL to 480 µg/mL were chosen for in vitro mammalian cell gene mutation tests in the absence and the presence of S-9.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
There was no indication for induced cytotoxicity in the absence and presence of S-9. In experiment I, the highest concentration plated for viability assessment (%RPE) was 120 µg/mL in the absence of S-9 and 480 µg/mL in the presence of S-9, which gave ~115.2% and ~72.6% RPE, respectively. In experiment II, the highest concentration plated for viability assessment (RPE) was 480 µg/mL in the absence and presence of S-9, which gave ~102% and ~82.6% RPE, respectively.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Experimental Result

Experiment I, 3h treatment	Treatment (µg/mL)	S9 mix	%RPE	MF^a
	Vehicle	-	100	10.8
	3.75	-	121	10.3
	7.5	-	116.8	8.9
	15	-	115.3	15.1
	30	-	117.1	30.7*
	30	-	110.7	19.8
	120	-	115.2	10.1
	EMS (600)	-	120.5	166.7*
	Vehicle	+	100	6.3
	7.5	+	84.9	5.3
	15	+	100	1
	30	+	96.5	3.9
	30	+	80.5	5.3
	120	+	91.1	16.1*
	240	+	95.6	17.9
	480	+	72.6	4.9
	DMBA (3)	+	79.6	123.9*
Experiment II, 3h treatment	Vehicle	-	100	17
	7.5	-	107.4	6.5
	15	-	106.6	9.5
	30	-	116.5	21.4
	30	-	88	31.6
	120	-	108.3	9.4
	240	-	98.7	14.1
	480	-	102	12.5
	EMS (600)	-	94.7	227.6*
	Vehicle	+	100	19.5
	7.5	+	100.8	31.5
	15	+	107.2	9.6
	30	+	110.3	7.3
	30	+	103.6	17.8
	120	+	98.4	39.7*
	240	+	98.8	5.9
	480	+	82.6	10.7
	DMBA (3)	+	67.6	327.8*

^a 6TG-resistant mutants/10⁶ viable cells 7 days after treatment

* statistically (p < 0.01) significant using the Kruskal-Wallis test

Conclusions:

It is concluded that the test substance did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study.

Executive summary:

In a GLP-compliant OECD 476 study the test substance was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in Chinese hamster lung V79 cells. The study consisted of a cytotoxicity Range-Finder experiment followed by two main mutation experiments, each conducted in the absence and presence of metabolic activation by 20-methylcholanthrene induced rat liver post-mitochondrial fraction (S-9). The test item was formulated in DMSO and dosed at 1% v/v. A 3 hour treatment incubation period was used for all experiments. The highest concentration tested which gave acceptable survival (measured by relative plating efficiency [RPE]) was 480 µg/mL (limit of solubility) in the absence of S-9, which gave ~110.4% RPE. Accordingly, for Experiment 1 six or seven concentrations ranging from 3.75 to 480 µg/mL were tested both in the absence and presence of S-9. Seven days after treatment, the highest concentration selected to determine viability and 6TG resistance was 120 µg/mL in the absence of S-9 and 480 µg/mL in the presence of S-9, which gave ~ 115.2 % and 72.6% RPE, respectively. In Experiment 2 the test article was tested in conc. range of 7.5 - 480 µg/mL both in the absence and presence of S-9. Seven days after treatment the highest concentration selected to determine viability and 6TG resistance was 480 µg/mL both in the absence and presence of S-9, which gave ~ 102% and ~ 82.6% RPE, respectively. Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies in negative control were consistent with the acceptable range and clear increases in mutant frequency were observed by the positive controls. The assay system was therefore considered to be both sensitive and valid.

In Experiment 1 in the absence of S-9 statistically significant increase in mean mutant frequency (MMF) was only observed at concentration of 30 µg/mL. At this level RPE was not reduced (RPE ~ 117.1% ). The increase in MMF was not greater than 3-fold above that of the concurrent vehicle control and this effect was not reproduced in independent Experiment 2. In the presence of S-9, statistically significant increase in mean mutant frequency was observed at concentration of 120 µg/mL only. Fold increases greater than 3-fold over the vehicle control was not observed and mutant frequency fall within the range of historical control (for solvent DMSO). Cytotoxicity (expressed in terms of %RPE at the end of treatment) was ~ 91.1 %.

In experiment 2 in the absence of S-9, exposure to concentrations up to 480 µg/mL for 3h resulted in a negative response. Cytotoxic effects of test item were not observed in the whole concentration range of 7.5 - 480 µg/mL. In the presence of S-9 statistically significant increase in mean mutant frequency was found at concentration of 120 µg/mL only. At this concentration the increase in MMF was not greater than 3-fold above that of the concurrent vehicle control. There was no indication for induced cytotoxicity. No evidence of an increase in MMF was observed at any of the other concentrations tested.

It is concluded that the test substance did not induce mutation at the hprt locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 July 2001 - 15 August 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-10 containing HEPES buffer. For cell growth and treatment in the absence of S9 mix, fetal bovine serum (10% v/v) was added. The medium used for treatment in the presence of S9 mix and for washing cultures before or after treatment, was serum free.
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced S9 Mix prepared from livers of Fischer rats

Test concentrations with justification for top dose:

625, 1250, 2500, and 5000 µg/mL
(1250 µg/ml was not evaluated for chromosomal aberrations)

Vehicle / solvent:

Dimethylsulphoxide (1%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9-mix: Cyclophosphamide (30, 40 µg/mL); Without S9-mix: Methyl-methanesulphonate (20, 30 µg/mL)

Details on test system and experimental conditions:

DURATION
- Exposure duration: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR: Colcemid (0.1 µg/mL; 2 hours before harvesting)

STAIN:
Three slides per culture were made. Slides were prepared by dropping the cell suspension on to clean, grease-free slides. The slides were stained with Giemsa, then made permanent by mounting coverslips with DPX mountant.

NUMBER OF REPLICATIONS:
Treatments with test item or vehicle control substances were performed on duplicate cell cultures.

NUMBER OF CELLS EVALUATED:
From 2 slides per culture, up to 50 metaphase cells per slide, a total of 100 metaphase cells per culture, were examined.

DETERMINATION OF CYTOTOXICITY
- Slides were examined for evidence of metaphase cells and signs of cellular necrosis.

Evaluation criteria:

Acceptance criteria:
The experiments in this study were deemed to be valid because they fulfilled the following criteria:
-There was no evidence of contamination.
-Cells in vehicle control cultures had normal growth.
-The results of vehicle and positive control cultures were typical.
-The test item had 3 acceptable dose levels for assessment.

Interpretation of clastogenicity:
The results for test item and positive control treated cultures are evaluated by comparison with the concurrent vehicle control cultures and with historical negative control data. A negative response was recorded if responses from the test item treated cultures are within the 95% confidence limits for the historical negative control data. The response at a single dose was classified as significant if the percent of aberrant cells is consistently greater than the 99% confidence limits for the historical negative control data or greater than double the frequency of an elevated vehicle or untreated control culture if appropriate. A test was positive if the response in at least one acceptable dose level is significant by the criterion described above. A test item was positive if Test 1 was positive, as described above or if one of the tests was positive and the other test gave indications of activity. These indications may be suspicious levels of aberrant cells (between 95% and 99% confidence limits). Experiments that met in part the criteria for a positive response, or marginally met all the criteria, were classed as inconclusive.

Interpretation of toxicity:
A dose level was considered to be toxic if the cell count was reduced to less than 50% of the mean vehicle control culture values or if consistent evidence of changes to cell morphology was observed.

Statistics:

Toxicity: from the cell counts, the number of cells recovered per culture, was calculated. This was then compared with the number of cells (mean of 2
cultures) recovered from the vehicle control cultures.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

clastogenic in the presence of S9 mix at precipitating concentration toxic to the cells

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With S9-mix: 2500 and 5000 µg/mL; without S9-mix: 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The test item did not change the color of the culture medium, therefore no pH measurements were made.
- Effects of osmolality: During the test, the osmotic pressure of selected concentrations of the test item was measured. The test article did not affect the osmolality of the culture medium, therefore no further measurements were made.
- Precipitation: 1250, 2500 and 5000 µg/mL (with S9-mix)

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control cultures had levels of structural and numerical aberrations within the 95% confidence limits of the historical negative control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9 mix, toxicity was noted in cultures treated with 2500 and 5000 µg/mL. The cultures treated with 5000 µg/mL had reduced cell counts (below 50% of the vehicle control cultures) and 2500 µg/mL was deemed toxic to the cultures from culture and slide observations. In the absence of S9-mix, reduced cell counts were observed in the cultures treated with 5000 µg/mL.

ADDITIONAL INFORMATION ON CLASTOGENICITY:
Due to the clearly positive responses in the first test it was not deemed necessary to carry out the second test or carry out an extra assessment of polyploidy. It was noted, however, that in the absence of S9-mix there was an increase in the number of polyploidy cells observed during the assessment for structural aberrations. This increase was in the cultures treated with 2500 and 5000 µg/mL. The results fulfill the criteria to judge the test article as a clastogen in the presence of S9-mix at precipitating concentrations that were deemed toxic to the cells.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results of the chromosomal aberration assay with Chinese Hamster Ovary cells in vitro in the absence and presence of S9-mix (6 h treatment, 24 h harvest).

 

In the presence of S9 mix, positive responses were observed in the cultures treated with 2500 and 5000 µg/ml. These were precipitating concentrations and were deemed toxic to the cells. In the absence of S9 mix all the cultures were within the 95% confidence limits of the historical negative control data for structural aberrations.

Test item	Concentration	Cell count data	Aberrant cells in %
	in µg/mL	in %	with gaps	without gaps
Exposure period 6h, fixation time 24h, without S9 mix
DMSO	1%	0.98; 1.02	0;0	0;0
Methyl- methanesulphonate	20	-	18	17
	30	-	37	36
Test substance	625	0.98	0	0
	625	0.84	0	0
	2500	0.79	0	0
	2500	0.62	0	0
	5000	0.25	1	1
	5000	0.19	0	0
Exposure period 6h, fixation time 24h, with S9 mix
DMSO		0.98; 1.02	0;0	0;0
Cyclophosphamide	30	-	9	7
	40	-	9	8
Test substance	625	0.93	1	1
	625	0.89	3	1
	2500	0.59	10	9
	2500	0.70	11	10
	5000	0.48	13	8
	5000	0.49	11	11

 

Conclusions:

The test material was clastogenic when tested with Chinese hamster ovary cells in vitro in the presence of S9 mix at precipitating concentrations that were deemed toxic to the cells.

Executive summary:

The test article was submitted for testing and evaluation of clastogenic potential. Chromosomal aberrations assays were performed with duplicate, Chinese hamster ovary (CHO) cell cultures. Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used. The test was conducted in the presence and absence of a post-mitochondrial supernatant fraction obtained from the livers of adult, male rats treated with Aroclor 1254 (S9) and a NADPH-generating system. Cultures, established approximately 20 h before testing, were treated for 6 h in both the presence and absence of S9 mix. Cultures were harvested at 24 h post treatment. The test substance was toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested to the maximum permitted concentration of 5000 µg/ml. Toxicity was noted at 2500 and 5000 µg/ml in the presence of S9 mix and at 5000 µg/ml in the absence of S9 mix. The test substance induced structural chromosomal aberrations in the presence of S9 mix. Based on the results of this study, it was concluded that the test item was clastogenic when tested with Chinese hamster ovary cells in vitro in the presence of S9 mix at precipitating concentrations that were deemed toxic to the cells.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 25 to February 8, 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

two plates per dose level instead of three, no repeat experiment performed

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: HIS operon
E. coli: TRP operon

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of phenobarbital and 5,6-Benzoflavone treated SD-rats.

Test concentrations with justification for top dose:

1, 5, 10, 50, 100, 500, 1000, and 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Untreated negative controls:

yes

Remarks:

Sterility controls (S9-Mix, test substance)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Benz[a]pyren, 2-Aminoanthracene, 9-Aminoacridine, 2-Nitrofluorene, 1-Ethyl-3-nitro-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Expression time (cells in growth medium): at least 48 hours at 37°C

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: Bacterial background growth

POSITIVE CONTROLS
Benz[a]pyren (BP; 5 µg/plate) was used in the presence of S9-mix with the Salmonella tester strains TA 98, TA 100, TA 1537, and TA 1538.
2-Aminoanthracene (2-AA) was used in the presence of S9-mix with the Salmonella tester strains TA 1535 (2 µg/plate) and E. coli WP2uvrA (80 µg/plate).
9-Aminoacridine (9-AA) was used in the absence of S9-mix with the Salmonella tester strain TA1537 (80 µg/plate)
2-Nitrofluorene (2-NF) was used in the absence of S9-mix with the Salmonella tester strains TA 98 (1 µg/plate) and TA 1538 (2 µg/plate).
1-Ethyl-3-nitro-nitrosoguanidine (ENNG) was used in the absence of S9-mix with the Salmonella tester strains TA100 (3 µg/plate), TA1535 (5 µg/plate) and E. coli WP2uvrA (2 µg/plate).

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Number (arithmetic mean) of colonies of mutants in experiments without microsomal activation.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538	E. coli WP2UVrA
0	30	125	14	11	27	21
1	32	136	13	11	37	17
5	33	116	10	11	27	17
10	29	135	17	10	34	8
50	30	115	18	11	26	19
100	32	125	16	11	34	18
500	35	121	14	12	34	16
1000	31	131	17	11	31	14
5000	28	120	13	11	32	11
ENNG
2						130
3		510
5			586
2-NF
1	376
2					130
9-AA
80				596

Table 2: Number (arithmetic mean) of colonies of mutants in experiments with microsomal activation.   

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538	E. coli WP2UVrA
0	34	130	20	41	40	16
1	39	127	18	48	44	19
5	49	136	18	37	38	16
10	49	122	19	39	41	16
50	45	117	17	36	48	25
100	51	132	17	40	46	21
500	45	125	13	32	42	25
1000	46	140	14	36	31	18
5000	40	127	14	36	43	18
2-AA
2			117
80						789
BP
5	306	882		151	115

Details on positive controls see under "details on test system and conditions"

 

Conclusions:

The test article did not cause increased numbers of colonies by reverse mutation, in either the presence or absence of a metabolic activating system compared to the control. Therefore, the test article was not found to be mutagenic in this Ames test.

Executive summary:

The test article's potential to cause point mutation in bacteria was investigated in an Ames test performed similar to OECD guideline 471. Salmonella typhimurium strains TA 100, TA 98, TA 1535, TA 1537, and TA 1538, and Escherichia coli strain WP2 uvrA, were exposed to the test material in the absence or presence of an in vitro metabolic activation system. All strains were tested by the preincubation method at concentrations of 1, 5, 10, 50, 100, 500, 1000 and 5000 µg/plate in duplicates. After an incubation period of 48 hours, the number of revertant colonies was evaluated. In this experiment, the test article did not cause an increase in the number of his+ or trp+ revertants compared to the control plates, either in the presence or absence of a metabolic activation system. Therefore, based on the results of this study, the test article was not found to be mutagenic in bacteria.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, acceptable for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 50, 100, 250, 500, 1000 and 1500 µg/ml

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Methyl methanesulfonate (MMS) was used at concentrations of 10 and 15 µg/ml of culture medium in the nonactivation assay while 20-methylcholanthrene (20-MCA) was used at concentrations ranging from 1.0-5.0 µg/ml.

Details on test system and experimental conditions:

Two independent mutation assays were conducted to assess the mutagenic potential of the test material.

Treatment Procedure
Cells from logarithmically growing stock cultures (maintained in an orbital shaker at 37C) were treated in individual 50 ml culture tubes. To these cells, 4 ml of either S9 mix or medium was added along with the test material. Following the addition of the test compounds, the tubes were incubated for approximately 4 hours at 37°C in a roller drum (approximately 25-35 orbits/minute). At the end of the incubation period, the cells were pelleted, rinsed with medium and resuspended. The tubes were returned to the roller drum and maintained at 37°C during a standard expression period of two days.

Toxicity Assay
This assay was conducted for selecting concentrations of the test material to be used in the gene mutation assay. The cells were treated with various concentrations of the test material (one replicate per dose) in the absence and presence of S9. At approximately 24 hours after treatment (day 1), the test cultures were counted. If the treated cells failed to multiply to a density of 4 x 10E05 on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2, cultures were again counted for cell density. From these cell counts, the following indices were calculated:
Day 1 SG = Suspension Growth over first day
Day 2 SG = Suspension Growth during second day
CSG = Cumulative Suspension Growth during first 2 days
Day 2 RSG (%) = Cumulative relative suspension growth over first two days

Gene Mutation Assay
Each dose level was set up in duplicate from the time of treatment until the completion of the assay with the exception of positive controls where only one replicate was used. At the end of treatment, cells were returned to the incubator for phenotypic expression. At 24 hours following treatment (day 1), the test cultures were counted and diluted to a concentration of approximately 3 x 10E05 cells/ml. If the treated cells failed to multiply to a density of 4 x 10E05 cells/ml on the first day following treatment, the culture was returned to the incubator without any dilution. On day 2 (48 hours following treatment) cultures were again counted and treatment levels with desired levels of toxicity were selected for cloning. Cultures with < 10% day 2 RSG were not cloned. A total sample size of 3 x 10E06 cells from each culture was suspended in cloning medium with trifluorothymidine (TFT) and plated into three petri dishes (100 mm), allowed to gel for approximately 15 minutes at 0-6°C, and returned to the incubator for approximately 13 days to allow for mutant colony formation. The cloning efficiency was determined by serially diluting the sample in cloning medium without 1 µg/ml TFT and then plating the cells into three petri dishes (100 mm) at a concentration of approximately 200 cells per dish. The dishes were returned to the incubator for approximately 13 days before counting the number of colonies per dish.
An image analyzer (LAI High-Resolution Colony Counting System, Loats Associates, Inc., Westminster, Maryland) was used to count and size colonies. The separation of small and large colonies was determined by inspection of colony sizing histograms of each culture. Mutant colonies form a bimodal distribution and the cutoff between the two distributions was set manually.
The parameter relative total growth (RTG) was used to determine the cytotoxicity of various treatments.
Initially, plates for cloning efficiency were counted and the RTG values calculated. Only those cultures with RTG values ≥ 10% were counted for mutants. However, one exception occurred in the positive control cultures in the presence of S9 in the initial gene mutation assay where the RTG values were less than 10% and the mutant plates were evaluated. These plates were deemed suitable for counting in the initial assay and the data was confirmed in the repeat assay using a lower concentration of the positive control.

Evaluation criteria:

Mutant frequencies were evaluated based upon biological significance criteria. The test chemical was considered positive when the conditions listed below were met:
a) the average mutant frequency in at least one dose level of the treated cultures (resulting in ≥ 10% relative total growth) was 90 x 10-6 above the average of the concurrent solvent controls (assuming these to be in the range of 35-140 x 10-6).
b) there was a positive dose related linear trend. This was tested using a linear trend test at alpha = 0.05, provided the above criteria was met.

The test material was considered negative in this assay if the following condition was met:
a) there was no evidence of increase in mutant frequency at RTG values ≥ 10%.

The test material was considered equivocal in this assay if the following conditions were met:
a) there was a significant increase in mutant frequency only at RTG values > 10% and < 20%.
b) there was no evidence of increase in mutant frequency at RTG values ≥ 20%.

Statistics:

The activation and non-activation assays were considered independent assays with their own solvent and positive controls. For an assay to be considered acceptable, (1) the mutation frequency of positive controls should have been significantly higher than the solvent controls and (2) the mutant frequency of the solvent controls should have been within 35-140 x 10-6. The solvent controls must have had an average absolute cloning efficiency between 65-120% and a cumulative suspension growth greater than eight.

Species / strain:

mouse lymphoma L5178Y cells

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:

In a preliminary toxicity assay, the test material was evaluated at concentrations ranging from 5.9 to 1500 µg/ml. In the absence of S9, little to no toxicity was observed with day 2 relative suspension growth (RSG) values ranging from 81.2 to 124.2%. In the presence of S9, the corresponding day 2 values ranged from 80.7 to 101.5%.

There were no biologically significant increases in the mutant frequencies in the test material-treated cultures compared to the solvent control cultures in either absence or presence of S9. The results of this in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay with triethanolamine indicate that under the conditions of this study, the test article was non-mutagenic when evaluated in the absence and presence of an externally supplied metabolic activation (S9) system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study which meets basic scientific principles, adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

Method: other: after Galloway, S.M. et al.: Environ. Mutagen. 7, 1-51

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

1510 - 4030 µg/ml (without S9)
6040 - 10100 µg/ml (with S9)
Doses were chosen for the aberration test based on a preliminary test of cell survival 24 hr after treatment.

Vehicle / solvent:

Water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: triethylenemielamine, mitomycin C. or cyclophosphamide

Species / strain:

Chinese hamster Ovary (CHO)

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:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study which meets basic scientific principles, adequate for assessment

Principles of method if other than guideline:

Method: other: after Galloway, S.M. et al.: Environ. Mutagen. 7, 1-51

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

100 - 2520 µg/ml (without S9)
330 - 10100 µg/ml (with S9)
In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ml or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

Vehicle / solvent:

Water, dimethyl suifoxide (DMSO), ethanol, or acetone, in that order of preference.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Species / strain:

Chinese hamster Ovary (CHO)

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:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study which meets basic scientific principles, adequate for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Method: other: after Haworth, S. et al.: Environ. Mutagen. 5, Suppl. 1, 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537 or TA97, TA98, TA100

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster metabolic activation

Test concentrations with justification for top dose:

up to 10 mg/plate, no further data available

Vehicle / solvent:

Distilled water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

TA98

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA97 and TA1537

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

all strains

Details on test system and experimental conditions:

Type: Ames test

Evaluation criteria:

No data

Statistics:

No data

Species / strain:

S. typhimurium, other: TA1535, TA1537 or TA97, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

but testes up to limit concentrations

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA1535, TA1537, TA97, TA98, TA100

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No induction of the micronucleus frequency in mouse bone marrow cells was observed for the triazine compound after treatment of mice with up to 2000 mg/kg body weight test substance.

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:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(adopted 1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: Hsd:ICR (CD-1)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD)
- Age at study initiation: 5-6 weeks
- Weight at arrival: 22-30 g
- Housing: 5 animals per cage in clear polycarbonate cages
- Diet: commercially available laboratory rodent diet (Altromin MT, Altromin, D-32770 Lage, Postfach 1120, Germany), ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: CMC (carboxymethyl cellulose, 0.5%)
- Concentration of test material in vehicle: 200 mg/mL; 100 mg/mL; 50 mg/mL
- Amount of vehicle: 10 mL/kg
- Batch No. (from BDH): 1072067

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of the test item, as received, were prepared immediately before use in carboxymethylcellulose 0.5%. Solutions were prepared on a weight/volume basis without connection for the displacement due to the volume of the test item.

Duration of treatment / exposure:

Not applicable.

Frequency of treatment:

Single treatment.

Post exposure period:

Control group: 24 and 48 h
500 mg/kg bw: 24 h
1000 mg/kg bw: 24 h
2000 mg/kg bw: 24 and 48 h
Positive control: 24 h

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Control group: 10 per sex
500 mg/kg bw: 5 per sex
1000 mg/kg bw: 5 per sex
2000 mg/kg bw: 10 per sex
Positive control: 5 per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin-C (batch no. 31K2504; SIGMA)
- Route of administration: oral, gavage
- Doses / concentrations: 3 mg/kg bw

Tissues and cell types examined:

Erythrocytes of femur bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A toxicity test was performed to aid in the selection of dose-levels. Groups of two male and two female mice were dosed by oral gavage with the test item at 2000, 1000 and 500 mg/kg bodyweight. The animals were observed for 48 hours and sacrificed. Scoring was performed on slides prepared from the femurs of animals.

DETAILS OF SLIDE PREPARATION:
Animals were sacrificed at appropriate sampling times and the femurs of animals were removed and bone marrow cells obtained by flushing with fetal calf serum. The cells were centrifuged and a concentrated suspension was prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. Where no depression of polychromatic erythrocytes was observed at least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.

Evaluation criteria:

The test item is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately. Where increases in the incidence of micronucleated PCE's are observed which are statistically significant, but fall within the range of negative control values within this laboratory, then concurrent and historical control data are used to demonstrate that these increases do not have biological significance.

Statistics:

Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups.
The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Reduced activity was observed in female animals on the day of the treatment and the day after. No other adverse reactions to treatment were noted. Treatment with the test item had no adverse effect on the proliferation of bone-marrow erythropoietic cells. Based on the above results, dose-levels of 500, 1000 and 2000 mg/kg bodyweight were selected for the Main assay.

RESULTS OF DEFINITIVE STUDY
No increases in the numbers of micronucleated PCE's were observed in any treatment group at any sampling time. Pronounced increases in the frequency of micronucleated PCE's were observed in the positive control group, indicating the correct functioning of the test system.
The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analyzed to evaluate the bone marrow cell toxicity. Based on these results, no inhibitory effect on erythropoietic cell division was observed in male or female animals at any sampling time.

Table 1: Incidence of micronucleated PCEs and ratio of PCE/NCE of the in-vivo bone marrow micronucleus study.

Treatment	Dose level (mg/kg bw)	Incidence of micronucleated PCEs				PCE/(PCE+NCE) % over the mean negative control value
	male/female	Mean	SE	range
24 hr sampling time
Vehicle	10 mL/kg	0.6	0.2	0	1.5	100
Test item	500	0.9	0.3	0	3.0	99
Test item	1000	0.5	0.2	0	1.5	101
Test item	2000	0.8	0.3	0	2.5	98
Mitomycin C	3	10.6***	1.4	6.0	17.0	102
48 hr sampling time
Vehicle	10 mL/kg	0.6	0.2	0	1.5	100
Test item	2000	0.8	0.3	0	3.0	98

***: Incidence significantly greater than control value at p < 0.001

Conclusions:

On the basis of the results obtained, it is concluded that the test article administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

Executive summary:

The test article's potential to cause chromosomal damage in vivo was investigated in a micronucleus test. Based on results obtained in a preliminary toxicity trial, dose-levels selected were 500, 1000 and 2000 mg/kg bodyweight for male and female animals. Hsd:ICR (CD-I) mice were dosed once by gavage with the vehicle alone (carboxymethylcellulose 0.5%), the test material in the vehicle at the selected dose-levels or with the positive control substance Mitomycin-C. Each group consisted of five male and five female animals with the exception of the control and high-dose group, which included an additional five animals of each sex per group. Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time. Bone-marrow smear slides were made and stained with May-Gruenwald and Giemsa stains. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei. The slides were coded prior to scoring. The results obtained at each sampling time were subjected to statistical analysis using a modified chi-squared test. Following treatment with the test substance, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose level, at any sampling time. Following treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCE's over the control values were observed, indicating the correct functioning of the test system. No clinical signs or depression of bone marrow erythropoietic cell division, were observed at any dose-level, at any sampling time. It is concluded that, under the reported experimental conditions, the test item administered by oral gavage at the selected dose-levels to male and female mice, does not induce micronuclei in the polychromatic erythrocytes.

Additional information

Additional information from genetic toxicity in vitro:

The substance is a salt which is a reaction product of an acid (triazine compound, CAS 80584-91-2) and a base (triethaonoleamine, TEA, CAS 102-71-6) that retain their ionic character. Under aqueous conditions it is expected to be present in its dissociated form. Therefore, the individual components of the salt are assessed separately.

CAS 80584-91-4:

The mutagenicity of the test substance was assessed in an Ames test performed with Salmonella strains TA 100, T A98, TA 1535, TA 1537, TA 1538 and in Escherichia coli WP2uvrA with up to 5000 μg per plate with and without microsomal activation. S9 was prepared from livers of phenobarbital and 5,6-Benzoflavone (BF) treated SD-rats. No evidence for a mutagenic effect was obtained in Salmonella strains TA 100, T A98, TA 1535, TA 1537, TA 1538 and in Escherichia coli WP2uvrA with up to 5000 μg per plate in experiments with and without microsomal activation.

In a GLP-compliant OECD 476 study the test substance was assayed for the ability to induce mutation at the HPRT locus in Chinese hamster lung V79 cells. The study consisted of a cytotoxicity Range-Finder experiment followed by two main mutation experiments, each conducted in the absence and presence of metabolic activation system at concentrations ranging from 3.75 to 480 µg/ml using an incubation period of three hours. The maximum concentration tested was limited by solubility. Mutant frequencies in negative controls were consistent with the acceptable range and clear increases in mutant frequency were observed by the positive controls. The assay system was therefore considered to be both sensitive and valid. Isolated cases of statistically significant increases in mean mutant frequency (MMF) were observed, however these cases did not reach the 3-fold threshold and were not dose dependent. There was no indication for induced cytotoxicity. It is concluded that the test substance did not induce mutation at the HPRT locus of V79 Chinese Hamster lung cells when tested under the conditions employed in this study.

A chromosomal aberration assay was performed with duplicate cultures using Chinese hamster ovary (CHO) cell cultures, according to OECD TG 473 and compliant with GLP (Ciba, 2002). Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were used as positive controls. Cultures were treated for 6 h in both the presence and absence of S9 mix and harvested at 24 h post treatment. Toxicity was noted at 2500 and 5000 μg/mL in the presence of S9 mix and at 5000 μg/mL in the absence of S9 mix. In the presence of S9 mix, positive responses were observed in the cultures treated with 2500 and 5000 μg/mL. These were precipitating concentrations and were deemed toxic to the cells. In the absence of S9 mix all the cultures were within the 95% confidence limits of the historical negative control data for structural aberrations. In the absence of S9 mix there was an increase in the number of polyploidy cells observed during the assessment for structural aberrations at 2500 and 5000 μg/mL. In conclusion, based on the result of this study, the test article was found clastogenic in vitro in the presence of S9 mix at precipitating concentrations that were deemed toxic to the cells.

The ability to cause chromosomal damage in vivo was investigated in a micronucleus test by oral gavage according to OECD TG 474 (adopted 1997) and GLP. Based on results obtained in a preliminary toxicity trial, dose-levels selected were 500, 1000 and 2000 mg/kg body weight for male and female animals. Mice were dosed once only with the vehicle alone (carboxymethylcellulose 0.5%), the test item at the selected dose-levels or with the positive control substance mitomycin-C (3 mg/kg bw). Five animals per sex from each group were sacrificed at the 24 hour sampling time. Additional animals (5 animals per sex of the control and high dose group) were sacrificed at the 48 hour sampling time. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei. Following treatment with the test substance, no statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose level, at any sampling time. No clinical signs or depression of bone marrow erythropoietic cell division were observed at any dose level, at any sampling time. Treatment with the positive control mitomycin-C caused the expected increase of micronuclei. On the basis of the results obtained, it is concluded that the test substance administered by oral gavage, does not induce micronuclei in the polychromatic erythrocytes of treated mice, under the reported experimental conditions.

CAS 102-71-6:

TEA was tested in an Ames reverse mutation assay using S. typhimurium strains TA 1535, TA 1537, TA 97, TA 98 and TA 100 at concentrations of up to 10000 µg/plate with and without metabolic activation. Treatment with TEA was not associated with reverse mutations in any of the strains tested (Mortelmans, 1986). In another bacterial mutation assay using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli strains WP2 and WP2 uvrA, TEA was tested at concentrations of up to 4000 µg/plate with and without metabolic activation. In this assay, TEA was not genotoxic in all the strains tested (Dean, 1985). TEA was also tested negative in a bacterial mutation study using S. typhimurium strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and E. coli strains WP2 and WP2 uvrA with and without metabolic activation at concentrations up to 2000 µg/plate (TSCATS, 1989). In a fourth mutation assay, using S. typhimurium strains TA 98 and TA 100 and E. coli strain WP2, TEA was tested at concentrations up to 20000 µg/plate with and without metabolic activation. In this assay, TEA was also tested negative (Innoue, 1982). Induction of chromosomal aberrations and sister chromatid exchanges was investigated in Chinese hamster ovary cells, exposed to concentrations up to 10100 µg/mL (which induced cytotoxicity). All tests were negative in the absence as well as the presence of metabolic activation (Galloway, 1987). TEA was also negative in the in vitro mouse lymphoma (L5178Y TK+/-) forward gene mutation assay (The Dow Chemical Company, 2010). Two independent assays at concentrations ranging from 50 to 1500 mg/ml in the absence and presence of an externally supplied metabolic activation (S9) system were performed. The highest concentration tested was the limit dose of 10 mM.

Based on the available information, IARC (2000) concluded that TEA was not mutagenic to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 or TA 1538 in the presence or absence of exogenous metabolic activation in a number of studies. TEA did not induce mutations in Escherichia coli WP2 uvrA and WP2 try- in the presence or absence of exogenous metabolic activation in two studies. In a single study, TEA was not mutagenic to Bacillus subtilis strains carrying uvrA or uvrA and polA mutations in the presence or absence of exogenous metabolic activation. However, when TEA was mixed with sodium mitrite, mutations were induced in this system without exogenous metabolic activation; this activity was lost in the presence of exogenous metabolic activation.

TEA did not induce gene conversion in Saccharomyces cerevisiae in the presence or absence of exogenous metabolic activation in one study. In a single study, sex-linked recessive lethal mutations were not induced in Drosophila melanogaster by treatment with TEA either by diet or injection. Unscheduled DNA synthesis was not induced in rat primary hepatocytes exposed to TEA in two studies. TEA did not induce sister chromatid exchanges in Chinese hamster ovary cells in either the presence or absence of exogenous metabolic activation. Chromosomal aberrations were not induced in rat liver cells, Chinese hamster lung cells or Chinese hamster ovary cells by in-vitro exposure to TEA. It did not induce cell transformation in Syrian hamster embryo cells.

Conclusion:

CAS 80584-91-4 was found negative in an Ames test and in an HPRT test with and without S9 mix. As a result, it is concluded that the test material has no potential to cause point mutations. In an in vitro chromosomal aberration study, CAS 80584-91-4 caused clear positive results in the presence of S9 mix when tested in Chinese hamster ovary (CHO) cell cultures at precipitating concentrations. However, these concentrations were deemed toxic to the cells and no clear conclusion regarding clastogenicity could be drawn. To further investigate the clastogenic potential, the test material was evaluated in vivo in a micronucleus test performed with mice. Here, no indication for clastogenic activity was reported even at the highest tested dose levels. Therefore, it is concluded that CAS 80584-91-4 has no potential to cause chromosomal damage and no classification regarding genotoxicity is required.

CAS 102-71-6 was similarly not negative in several Ames studies and in a mouse lymphoma assay, therefore no potential for point mutation is evident. TEA was also negative in various assays addressing chromosomal damage.

Since both components of the salt do not harbor any genotoxic potential, the salt itself is therefore considered to be of no concern in regard to genotoxicity.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance does not need to be classified and labelled under Regulation (EC) No 1272/2008.