1,1'-[(6-phenyl-1,3,5-triazine-2,4-diyl)diimino]bisanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- HPRT, OECD 476 and GLP: negative

- Ames, OECD 471 and GLP: positive with TA98 in the presence and absence of S9

- Ames II with highly purified test material: negative

- In vivo micronucleus study in hamsters: negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: modified OECD 471 protocol (Ames II), non-GLP

Principles of method if other than guideline:

Liquid fluctuation Ames II test with and without liver S9 mix from induced male Wistar rats.
The test method is used to evaluate the mutagenic potential based on the ability to induce point mutations in selected loci of several strains of Salmonella typhimurium in a modified version of the Ames test (OECD 471), designated Ames II Assay (microtiter version), both with and without the addition of a metabolizing system (S9 mix) obtained from liver from induced rats.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

other: TA 98, TA Mix (TA 7001 - TA 7006)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix

Test concentrations with justification for top dose:

O; 4; 20; 100; 500; 2500 and 5000 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (with S9) and 2-nitrofluorene plus 4-nitroquinoline- N-oxide (without S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Bacteria from overnight cultures showing an optical density of at least 2.0 (measured at a wavelength of 600 nm) were used. 5 ml of the overnight cultures were added to tubes containing 25 ml Ames II Exposure medium and were gently mixed. After thorough pipetting bactiera suspension, test substance (or vehicle or control) and S9 mix (if applicable) were added. The 24-well plates were incubated at 37°C with shaking at 250 rpm for about 90 minutes. After this incubation period, 2.8 ml Ames II Reversion indicator medium (containing bromocresol purple) was pipetted to each well of the 24-well plate. The contents of each well of the 24-well plates were distributed in 50 μl aliquots over 48 wells of a 348-well Revertant Colony Selection plate (RCSP). The plates were sealed in plastic bags and incubated at 37°C in the dark. After 48 hours incubation each 48-well section of the RCSP were scored and the number of positive wells (yellow= high number of his+ revertants) were counted.

NUMBER OF REPLICATIONS: 48 wells in triplicate plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: beckground lawn, decrease in the number of yellow wells

Evaluation criteria:

Evaluation was performed by the following comparisons/calculation:
- An increase in the mean number of positive wells in dose groups was compared to the mean value of the concurrent negative control (Evaluation factor 1 F).
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of the actual experiment (Evaluation factor 2 F). The baseline was derived from the mean spontaneous revertant number plus the value of standard deviation (mean+ SD) from the distribution of spontaneous data.
- An increase in the mean of revertant wells in dose groups was calculated on the basis of the baseline data of an experimental run (Evaluation factor 3 F). A run consists of a variable number of experiments generally testing different test substances together each using the same vehicle control. This leads to an accumulation of replicates for negative controls which was used to calculate the mean spontaneous reversion number for each run.
A test substance is considered mutagenic in this test system if more than a doubling of Evaluation factor 3 F is observed in at least one test group. This finding should be dose-dependent and/or reproducible.

Species / strain:

other: TA 98, TA Mix (TA 7001 - TA 7006)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

An increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

No bacteriotoxic effect (clearing of the background lawn, decrease in the number of yellow wells) was observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At 500 μg/ml and above

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

SUMMARY OF RESULTS

TA98 without S9 mix

DOSE [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^C	SD	1F	2F	3F
DMSO		0	0	1	0.3	1.0	0.47	1.0	0.7	0.5
4		1	0	1	0.7		0.47	0.7	0.5	0.3
20		0	1	0	0.3		0.47	0.3	0.2	0.2
100		2	1	1	1.3		0.47	1.3	0.9	0.6
500	p	3	4	2	3.0		0.82	3.0	2.0	1.4
2500	p	1	1	2	1.3		0.47	1.3	0.9	0.6
5000	p	3	1	2	2.0		0.82	2.0	1.4	1.0
4-NQO+2-NF		43	41	47	43.7		2.49	43.7	29.7	20.9

TA98 with S9 mix

DOSE [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^C	SD	1F	2F	3F
DMSO		0	1	1	0.7	1.0	0.47	1.0	0.7	0.4
4		3	1	1	1.7		0.94	1.7	1.1	0.6
20		0	2	3	1.7		1.25	1.7	1.1	0.6
100		0	5	1	2.0		2.16	2.0	1.4	0.8
500	p	5	3	1	3.0		1.63	3.0	2.0	1.1
2500	p	5	1	1	2.3		1.89	2.3	1.6	0.9
5000	p	4	1	2	2.3		1.25	2.3	1.6	0.9
2-AA		48	48	48	48.0		0.00	48.0	32.6	18.4

TA Mix without S9

DOSE [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^C	SD	1F	2F	3F
DMSO		1	1	0	0.7	1.0	0.47	1.0	0.7	0.8
4		0	0	0	0.0		0.00	0.0	0.0	0.0
20		1	0	2	1.0		0.82	1.0	0.7	0.8
100		0	0	0	0.0		0.00	0.0	0.0	0.0
500	p	0	0	1	0.3		0.47	0.3	0.2	0.3
2500	p	1	0	0	0.3		0.47	0.3	0.2	0.3
5000	p	0	1	0	0.3		0.47	0.3	0.2	0.3
4-NQO+2-NF		46	47	46	46.3		0.47	46.30	31.50	37.4

TA Mix with S9

DOSE [µg/ml]	REM	RCSP 1	RCSP 2	RCSP 3	MEAN	MEAN^C	SD	1F	2F	3F
DMSO		0	1	2	1	1	0.82	1.0	0.6	0.8
4		0	0	0	0.0		0.00	0.0	0.0	0.0
20		1	0	0	0.3		0.47	0.3	0.2	0.3
100		2	2	1	1.7		0.47	1.7	0.9	1.3
500	p	0	1	0	0.3		0.47	0.3	0.2	0.3
2500	p	0	0	0	0.0		0.00	0.0	0.0	0.0
5000	p	2	0	0	0.7		0.94	0.7	0.4	0.5
2-AA		19	20	23	20.7		1.70	20.7	11.4	15.5

REM = Remarks

P = Precipitation

RCSP = Revertant Colony Selection Plate (384-Well)

SD = Standard Deviation

MEAN = Mean of Replicates

MEANC = Mean corrected : < 1 = 1

F = Factor

1F = Based on Meanc

2F = Baseline Data I Based on Mean^c + SD

3F = Baseline Data I Based on Mean + SD of a Run

4-NQO + 2-NF = 4-nitroquinoline: 5 μg/ml + 2-nitroflourene 100 μg/ml

2-AA = 2-aminoanthracene: 125 μg/ml

Conclusions:

Under the experimental conditions of this study, the test substance is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation.

Reference 2

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

Study period:

24 November 2014 - 03 February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 (incl. QA statement)

Remarks:

Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 µg/mL) and amphotericin B (1%).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for spontaneus mutant frequency: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Experiment I, with and without S9: 0.41; 1.2; 3.7; 11.1; 33.3; (100) µg/ml
Experiment II, with and without S9: (0.5); 1.0; 3.0; 10; 80; 830 µg/ml
Numbers in parantheses: these cultures were discontinued.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Remarks:

with S9: DMBA, 2.2 µg/mL; without S9: EMS, 150 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-16 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the correspon¬ding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance was considered together.

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
- Precipitation: Precipitation of the test item was observed in the first experiment at 11.1 µg/mL and above with and without metabolic activation. In experiment II precipitation was noted at 80 and 830 µg/mL with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hours of treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) prior to removal to the test item. Precipitation occurred at 6.5 µg/mL and above after 4 hours treatment with and without metabolic activation. There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item. The dose range of the first experiment was set according to precipitation observed in the pre-experiment. A rather wide dose range was used in the second experiment to include soluble concentrations as well as the maximum possible concentration of 830 µg/mL.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Summary of Results

	conc. (µg/ml)	P	S9 Mix	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor	relative cloning efficiency I (%)	relative cell density (%)	relative cloning efficiency II (%)	mutant colonies / 10^6cells	induction factor
Experiment I / 4 h treatment				culture I					culture II
Solvent control with DMSO			-	100	100	100	6	1	100	100	100	7.4	1
Positive control (EMS)	150		-	99.9	101	101.8	93.8	15.5	97.9	89.4	100.3	96	13
Test item	0.41		-	99.2	95.5	100.2	11.4	1.9	99.9	86.8	103.4	14.1	1.9
Test item	1.2		-	99.5	107.1	103.3	18.2	3	95.8	98.2	103	9.1	1.2
Test item	3.7		-	96.9	105	103.8	20	3.3	100	92	105.8	6.7	0.9
Test item	11.1	P	-	101.4	96.9	101.4	14.9	2.5	101.1	88.7	101.6	7.7	1
Test item	33.3	P	-	97	89	103	7.3	1.2	98	84.5	105.2	12.9	1.7
Test item	100	P	-	99.9	culture was not continued#				96.8	culture was not continued#
Solvent control with DMSO			+	100	100	100	10.1	1	100	100	100	20.8	1
Positive control (DMBA)	2.2		+	96	95.2	96	282.3	27.8	97.5	81.4	77.9	345.3	16.6
Test item	0.41		+	92.7	107.7	97.5	10.6	1	95.1	84.7	80.8	27.1	1.3
Test item	1.2		+	96.2	74.7	103.6	11.4	1.1	100.4	79.4	93.8	28.6	1.4
Test item	3.7		+	89.6	96.3	93.8	3.3	0.3	97.5	51.6	97.5	13.3	0.6
Test item	11.1	P	+	94.5	84	75.2	12.4	1.2	96.8	45.6	38.4	0	0
Test item	33.3	P	+	91.8	87.2	85.8	12.3	1.2	99.2	71.1	94.4	21	1
Test item	100	P	+	93	culture was not continued#				99.5	culture was not continued#
Experiment II / 4 h treatment				culture I					culture II
Solvent control with DMSO			-	100	100	100	9.3	1	100	100	100	28.6	1
Positive control (EMS)	150		-	96.7	92.5	101.7	165.3	17.7	96.9	95.5	88.1	207.1	7.2
Test item	0.5		-	100.8	92.4	culture was not continued##			96.9	86.6	culture was not continued##
Test item	1		-	102.7	105.1	103.6	5.9	0.6	97.1	85.5	114.2	20.9	0.7
Test item	3		-	102.9	117.7	101.3	18.8	2	100.7	91.9	94.7	20.7	0.7
Test item	10		-	104.1	96.6	101.4	16.4	1.8	99.3	111.6	96.6	17.1	0.6
Test item	80	P	-	104	93.8	104.7	5.7	0.6	93.3	106.1	97	24.5	0.9
Test item	830	P	-	110	97.3	105.9	18.3	2	101.6	104.5	88.8	39.5	1.4
Solvent control with DMSO			+	100	100	100	14.5	1	100	100	100	9.3	1
Positive control (DMBA)	2.2		+	92.4	101	102.4	367.6	25.3	96	96.5	93.2	390.3	42.2
Test item	0.5		+	100.7	94.3	culture was not continued##			93.5	108.6	culture was not continued##
Test item	1		+	87.4	104.6	103.2	9	0.6	97.1	96.1	110.8	10.7	1.2
Test item	3		+	104.4	108.3	102.3	5.3	0.4	92.4	115.6	111.6	9.6	1
Test item	10		+	108.1	109.1	99.1	12.3	0.8	99.7	114.9	93.7	6	0.6
Test item	80	P	+	93.9	104.7	109	18.2	1.3	101.9	111.7	94	18	1.9
Test item	830	P	+	108.7	155.6	106.3	21	1.4	94.1	145.4	96.1	17.6	1.9

P = Precipitation visible at the end of treatment

# culture was not continued to avoid analysis of too many precipitating concentrations

## culture was not continued as a minimum of only four analysable concentrations is required

Conclusions:

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Executive summary:

In a GLP-compliant genotoxicity study according to OECD guideline 476 the test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The main experiments were performed with and without liver microsomal activation and a treatment period of 4 hours. The highest concentration of 830 µg/mL was limited by the solubility of the test item in organic solvents. Precipitation in aqueous media occurred at lower concentrations. The test item was dissolved in DMSO. The tested concentrations of the main experiments ranged from 0.41 to 830 µg/ml. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Two MNT and two SCE studies produced all negative results.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

Difference in treatment schedule (12 week treatment, 3 times a week), No positive control

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: female: 23-30 g, male: 22-34 g
- Diet: NAFAG No. 924
- Water: Tap water ad libitum
- Acclimation period: Air conditioned room

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24 °C
- Humidity (%): 50-72 %
- Photoperiod: Illuminated for 12 hours

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Polyethylene glycole 400
- Concentration of test material in vehicle: 250, 500, 1000 and 2000 mg/kg in 20 mL/kg PEG 400

Details on exposure:

Treatment schedule: The preparation was administered orally to groups of 6 female and 6 male animals each, thrice weekly for twelve weeks. Six hours after the last application the animals were sacrificed by dislocation of the cervical vertebra.

Duration of treatment / exposure:

12 weeks

Frequency of treatment:

thrice weekly

Post exposure period:

no

Dose / conc.:

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

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:

6 males
6 females

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

Bone marrow from the shafts of both femurs.
Cell types examined: Single Jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronucleiin leuocpoietic cells, polyploid cells.

Details of tissue and slide preparation:

PREPARATION OF BONE MARROW:
The animals were sacrificed six hours after the last application. From the bone marrow smears were made.Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were airdried. Three hours later, the slides were stained in undiluted May-Griinwald solution for 2 min then in May-Griinwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol, 55% for 5-8 sec and washed off twice in water, they are left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying the slides were cleared in Xylol and mounted in Eukitt.

DETAILS OF SLIDE PREPARATION:
The slides of three female and three male animals per group were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: a) Single Jolly bodies, b) fragments of nuclei in erythrocytes, c) micronuclei in erythroblasts, d) micronucleiin leucopoietic cells, e) polyploid cells.

Statistics:

The significance of difference was assessed by X^2 test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

Of the twelve animals in the control group one female and one male animal died during the seventh week of the experiment. Of the animals treated with the lowest dose (250 mg/kg) one female and one male animal died during the second week of treatment. In the group treated with 500 mg/kg one male animal of the twelve animals died by the sixth week. Of the twelve animals in the group given 1000 mg/kg of the test substance all survived. In the group treated with the highest dose (2000 mg/kg) two male animals died during the second and the sixth week respectively of the treatment and one male animal died during the seventh week. In all dosage groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control.

	Number of animals	Sex of females	Single Jolly bodies	Fragments of nuclei in erythrocytes	Micronuclei in erythroblasts	Micronuclei in leucopoietic cells	Polyploid cells	Total
Control	1	m						0.0
(PEG 400)	2	m	0.1					0.1
	3	m	0.1					0.1
	4	f						0.0
	5	f	0.2					0.2
	6	f	0.1					0.1
Test item	1	m	0.1		0.1			0.2
(250 mg/kg)	2	m	0.1					0.1
	3	m						0.0
	4	f			0.3			0.3
	5	f	0.1					0.1
	6	f	0.1					0.1
Test item	1	m						0.0
(500 mg/kg)	2	m	0.1					0.1
	3	m	0.2					0.2
	4	f						0.0
	5	f	0.1			0.1		0.2
Test item	6	f						0.0
(1000 mg/kg)	1	m						0.0
	2	m	0.1					0.1
	3	m	0.2					0.2
	4	f	0.1					0.1
	5	f						0.0
	6	f	0.1				0.1	0.2
Test item	1	m						0.0
(2000 mg/kg)	2	m				0.1		0.1
	3	m	0.1					0.1
	4	f				0.1		0.1
	5	f	0.4					0.4
	6	f	0.1					0.1

Conclusions:

The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

AMES-Test

In a GLP compliant study following OECD guideline 471, the test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium TA 1535, TA100, TA1537, TA 98 and Escherichia coli WP2 uvrA, in a reverse mutation assay at dose levels of 33 μg - 5000 μg/plate. A standard plate test (SPT) was performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 333 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. According to the results of the present study, the test substance led to a dose dependent increase in the number of his+ revertants with the strain TA 98 both with and without S9 mix. The increase of revertants was reproducible in two experiments carried out independently of each other. Based on the recent assessment criteria the test substance has to be considered positive. In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. Thus, under the experimental conditions chosen here, it is concluded that the test item is a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation (BASF, 2015).

This finding was confirmed in an older supporting study. This study was performed with the following concentrations with and without microsomal activation: 25, 75, 225, 675 and 2025 µg/0.1 mL. In the experiments without and with microsomal activation, the number of back-mutant colonies of Strain TA 98 was significantly greater after treatment with the test substance than in the controls. A slight increase in the number of back-mutant colonies was also observed in the experiments without microsomal activation on Strain TA 1535 and TA 1537. The test item thus exerted a mutagenic potential in this test system (Ciba-Geigy, 1979).

A third Ames test is available yielding negative results. In this study, the test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies (RCC, 2007).

The positive results described above are most likely triggered by an unknown impurity, since the third available study using test material from a different batch produce a clear negative result. To confirm this hypothesis, the test article batch that caused a positive result in the latest Ames test was further purified and a follow-up Ames II study was performed with this highly purified test material. To ensure comparability of the Ames II findings, the test material of the standard purity (before the additional purification step) was also investigated in an Ames II study.

In the Ames II study with the standard purity test material, two vehicles were used (ultrapure water and DMSO) in order to investigate if the positive finding observed before could be related to chemical interaction of the unknown impurity present in the test item with DMSO. According to the results of this study, the test item led to an evident and partly dose-dependent increase in the number of revertant wells with the strain TA 98 with and without S9 mix using vehicle DMSO and only with S9 mix using vehicle ultrapure water. Thus, under the experimental conditions of this study, the test substance is a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation. The magnitude of the positive finding in tester strain TA98 with S9 mix was similar for both vehicles, therefore the positive reaction observed is apparently independent from the vehicle used (BASF, 2015).

In the follow-up Ames II study using the highly purified test material of the same batch, only DMSO was used as vehicle. According to the results of this study, an increase in the number of positive wells (his+ revertants) was not observed either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the purified test substance is not a mutagenic substance in the Ames II Assay (Salmonella typhimurium reverse mutation assay) in the absence and the presence of metabolic activation. It can therefore be concluded, that the positive results obtained previously were indeed due to an unknown impurity. The additional purification step removed this unknown impurity and a positive result was no longer obtained (BASF 2016).

This purification step will be implemented into production and all future batches produced will be characterized by this increased purity that does not lead to a positive result in the Ames test.

HPRT-Test

A GLP-compliant study following OECD guideline 476 was performed to assess the test item's potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The study was performed in two independent experiments, using identical experimental procedures. In both experiments the treatment period was 4 hours with and without metabolic activation. The main experiments were evaluated at concentration range of 0.41 - 830 µg/ml. Precipitation of the test item was observed in the first experiment at 11.1 µg/mL and above with and without metabolic activation. In experiment II precipitation was noted at 80 and 830 µg/mL with and without metabolic activation. No relevant cytotoxic effect indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures was observed up to the highest concentration of both experiments with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of the first experiment without metabolic activation at 1.2 and 3.7 µg/mL. This effect however, was based upon a rather low solvent control of 6.0 mutant colonies/106 cells and thus, biologically irrelevant. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely determined in the second culture of the second experiment without metabolic activation. However, the trend was judged as biologically irrelevant as the mutation frequency did not exceed the threshold described above or the historical range of solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 6.0 up to 28.6 mutants per 106 cells; the range of the groups treated with the test item was from 0.0 up to 39.5 mutants per 106 cells. EMS (150 µg/mL) and DMBA (2.2 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay (Harlan, 2015).

Genetic toxicity in vivo

The potential of the test item to cause genotoxicity was observed in two in vivo nucleus anomaly tests. In the key study the test item was administered by gavage thrice weekly for twelve weeks to Chinese hamsters at dose levels of 250, 500, 1000 and 2000 mg/kg. The animals were sacrificed six hours after the last application. From the bone marrow smears were made. The bone marrow smears from animals treated with various doses of the test item showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item (Ciba-Geigy, 1981)

In the supporting study Chinese hamsters were treated by gavage with one daily application on 2 consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The bone marrow smears from animals treated with various doses of the test item showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg) yielded 9.4% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (PEG 400) alone (Ciba-Geigy, 1980).

Two sister chromatid exchange assays are available for the test article. In the first assay (Ciba-Geigy, 1980), a positive result was reported in the highest dose group. However, this positive result was mainly triggered by the result obtained in one animal; with the other animals of this dose group the responses were similar to the control group. Therefore, this one finding might have been a chance finding and a confirmation experiment was performed with more dose groups and double animal numbers. This repeat assay clearly demonstrated that oral treatment with the test item does not trigger an increase in sister chromatid exchanges (Ciba-Geigy, 1983).

Justification for classification or non-classification

Positive findings in two Ames studies were initially available for the test material, contradicted by a negative result in an Ames test using a different test material batch. It could be shown that these positive findings were due to an unknown impurity. After further purification of the positive tested batch, the positive finding could no longer be reproduced. It can therefore be concluded that the additional purification step has removed the unknown impurity responsible for the positive findings. Since this additional purification step will be implemented into the standard production of the test article, there is no longer any concern regarding the positive Ames findings previously reported.

The test article was further negative in an HPRT study, negative in two in vivo micronucleus assays, and negative when tested for sister chromatid exchanges. Based on the available data, classification for genotoxicity is not warranted.