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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

EC Number:

279-505-5

EC Name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

Cas Number:

80584-91-4

Molecular formula:

C21H36N6O6

IUPAC Name:

6,6',6''-(1,3,5-triazine-2,4,6-triyltriimino)trihexanoic acid

Details on test material:

- Physical state: White powder
- Storage condition of test material: Ambient temperature (15- 25°C)

Method

Target gene:

hprt

Species / strain

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

Controls

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.

Results and discussion

Test results

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.