2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies generally conducted to recognised training guidelines with GLP

Link to relevant study records

Reference

Reference 1

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:

2006-02-07, 2006-06-06

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)

Version / remarks:

(1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 complete culture medium supplemented with 15 % horse serum (HS), 100 U/100 pg/mL Penicillin/Streptomycin, 220 pg/mL Sodium-Pyruvate, and 1.25 U/mL Amphotericin used as antifungal.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of liver from Wistar rats (HanIbm) induced with phenobarbital and beta-naphthoflavone, mixed with a solution of co-factors.

Test concentrations with justification for top dose:

The cultures were evaluated at the following concentrations of the test item:
experiment I:
without S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL
with S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

experiment II:
without S9 Mix: 1.3*; 2.5*; 5.0; 10.0; 20.0; 30.0; and 40.0 µg/mL
with S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

experiment III:
without S9 Mix: 5.0*; 10.0; 20.0; 40.0; 80.0; and 160 µg/mL

Following the expression phase of 72 hours the cultures marked with an asterisk were not continued since a minimum of only four analysable concentrations is required by the guidelines.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: The test substance is insoluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 19.5 µg/mL (4 h); 13.0 µg/mL (24 h)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation Migrated to IUCLID6: 4.5µg/mL

Details on test system and experimental conditions:

PRE-TEST:
A pre-test was performed in order to determine the concentration range of the main experiments.
1xE+7 cells were exposed to each concentration of the test item for 4 and 24 hours without and 4 hours with metabolic activation. During the 4 h treatment period the serum concentration was reduced from 15 % to 3 %. Following treatment the cells were washed and finally resuspended in 30 mL complete culture medium for a 2-day growth period. The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector (Mutation Research 31, 17-29, 1975).
Test item concentrations between 5.1 and 650 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Relevant toxic effects were observed at 325 µg/mL and above in the absence and at 81.3 µg/mL and above in the presence of metabolic activation (4 h treatment). Following the 24 h treatment without metabolic activation toxic effects occurred at 40.6 µg/mL and above.

MAIN TEST:
Experiment I
The treatment duration in the first experiment was 4 hours with and without metabolic activation.
Experiment II
The cells were treated with test item for 4 h with and for 24 h without metabolic activation. The experimental part without metabolic activation was terminated prior to the generation of any data on mutagenicity due to exceedingly severe toxic effects even at lower concentrations. Therefore, this experimental part was repeated as experiment IIA in a lower concentration range. The data of experiment IIA are reported as experiment II without metabolic activation.
Experiment III
This experiment was performed to verify a minor increase of the mutation frequency without metabolic activation in experiment I. Experiment III was performed in the absence of metabolic activation with a treatment time of 4 hours.

All experiments were performed in duplicate. Expression time of cells in growth medium was 48 - 72 h and selection time ranged from 10 to 15 days. The selection agent was Trifluorothymidine (TFT; 5 µg/mL). The number of replications was 2 and 2.0 cells/well in a 96-well microtiter plate were evaluated.

DETERMINATION OF CYTOTOXICITY:
Cytotoxicity was assessed using relative total growth and cloning efficiency 1= ln(number of empty wells on plates/192)/cells seeded per well.

Evaluation criteria:

EVALUATION OF RESULTS:
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the mutation frequency. Such an evaluation may be considered independently of the enhancement factor for induced mutants.
A positive response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative and solvent controls and the mutation rates of all negative and solvent controls of this study are taken into consideration.
Results of test groups are rejected if the relative total growth, the relative suspension growth, and/or cloning efficiency 1 is less than 10 % of the solvent control or the cloning efficiency 2 after the expression period is less than 20 %.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies, clastogenic effects are indicated.

ACCEPTABILITY CRITERIA:
A mutation assay is considered acceptable if it meets the following criteria:
All plates, from either the cloning efficiency 2 (ln(mean number of empty wells per plate / 96) / cells seeded per well) or the TFT resistance-testing portion of the experiment are analysable.
- The absolute cloning efficiency 2 of the negative and/or solvent controls is > 0.5 (50%).
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range of our historical control data
- The positive controls induce significant (at least 2-fold) increases in the mutant frequencies. The cloning efficiencies and the relative total growth are greater than 10% of the concurrent vehicle control group.

Statistics:

A linear regression was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT statistics software. The number of mutant colonies obtained for the groups treated with the test item was 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 and statistical significance should be considered together.
The data generated at the second precipitating concentration (160 µg/mL) are not included in the statistical evaluation.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A dose dependency of mutation frequency (Exp. I and II) was not considered biologically relevant. They either were not reproduced in the parallel culture (Exp. I) or absolute values of mutation frequency remained within the historical controls.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxic effects were seen in experiment II at 30 and 40 µg/ml without and 160 µg/ml with S9-mix and in experiment III (without S9-mix) at 80 µg/ml and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
Precipitation was seen at 80 µg/mL and above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the mouse lymphoma assay precipitation is problematic since the cells grow in suspension. At the end of the treatment, the cells are separated from the test item by centrifugation. A precipitate is likely to contaminate the cell-pellet at the centrifugation step. Thus, arbitrary amounts of test item are carried over to the following steps of the assay leading to non-defined exposure times of up to 48 hours to selection. Toxic or seemingly mutagenic artefacts are likely to occur under these conditions.

General Remarks

Toxicity

Relevant toxic effects, indicated by relative cloning efficiency 1 or a relative total growth of less than 50 % in both parallel cultures, were observed in the second experiment at 30 and 40 μg/mL without metabolic activation and at 160 μg/mL with metabolic activation. In the third experiment relevant toxic effects were noted in the precipitating range at 80 μg/mL and above.

In the experimental parts using 4 hour treatment, the dose range was limited by the solubility of the test item. Two precipitating concentrations were analyased. In the experimental part using continuous treatment for 24 hours without metabolic activation (experiment II), cytotoxic effects were dose limiting.

 

Mutagenicity

The data with metabolic activation do not indicate a possible mutagenic potential of the test item. Generally elevated but not dose dependent levels of the mutation frequency observed in the first culture of the first experiment with metabolic activation were based on the relatively low solvent control. Compared to the corresponding negative control no relevant increase occurred.

The data generated in the absence of metabolic activation with a treatment period of 4 hours showed an isolated increase of the mutation frequency at the maximum concentration of 160 μg/mL in the presence of precipitation.

Following 24 hours of treatment, no increase at all was noted even at severely cytotoxic concentrations in the soluble range. The increase at precipitating concentrations occurred in the second culture of the first experiment without metabolic activation and in the first culture of the third experiment. The threshold of twice the colony count of the corresponding solvent control was exceeded. No comparable increase occurred in the parallel cultures under identical conditions. Both of the increased levels of the mutation frequency are judged as biologically irrelevant precipitation artifacts.

 

Controls

In this study the range of the negative and solvent controls was from 34 up to 209 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 32 up to 538 mutant colonies per 10⁶ cells. Both solvent controls did not quite reach the historical range of solvent controls in the second experiment without metabolic activation. However, this effect was judged as biologically irrelevant fluctuation since both corresponding negative controls remained within the range of historical negative controls.

 

Statistics

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT® statistics software. The data generated at the second precipitating concentration of 160 μg/mL are not included in the statistical evaluation. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in experiment I culture I with and without metabolic activation. In experiment II such a trend was observed in both cultures without metabolic activation. However, the significant trends mentioned above were not considered biologically relevant since they either were not reproduced in the parallel culture (experiment I) or the absolute values of the mutation frequency remained within the historical range of negative and solvent controls (experiment II).

Table 2. Summary of results.

	conc. μg/mL	S9 mix	relative cloning efficiency 1	relative total growth	mutant colonies/ 10^6 cells	induction factor	relative cloning efficiency 1	relative total growth	mutant colonies/ 10^6 cells	induction factor
Column	1	2	3	4	5	6	7	8	9	10
Experiment 1/ 4 h treatment			culture I				culture II
Neg. control wtth medium		-	100.0	100.0	148		100.0	100.0	103
Solv. control with DMSO		-	100.0	100.0	165	1.0	100.0	100.0	181	1.0
Pos. Control with MMS	19.5	-	67.7	25.9	703	4.8	49.2	25.6	449	4.3
Test item	5.0	-	112.8	culture was not continued*			106.6	culture was not continued*
Test item	10.0	-	108.2	151.2	90	0.5	101.6	61.3	387	2.1
Test item	20.0	-	106.0	112.5	117	0.7	79.8	55.3	261	1.4
Test item	40.0	-	94.6	107.5	169	1.0	104.9	70.6	262	1.4
Test item	80.0 (p)	-	71.7	85.8	217	1.3	122.4	60.1	292	1.6
Test Item	160.0 (p)	-	56.8	72.5	280	1.7	81.0	44.6	538	3.0
Neg. control with medium		+	100.0	100.0	209		100.0	100.0	120
Solv. control with DMSO		+	100.0	100.0	106	1.0	100.0	100.0	66	1.0
Pos. control with CPA	4.5	+	49.9	67.8	703	3.4	57.0	69.8	336	2.8
Test item	5.0	+	100.0	culture was not continued*			73.6	culture was not continued*
Test item	10.0	+	83.9	102.1	193	1.8	63.0	156.5	43	0.7
Test item	20.0	+	87.0	84.6	230	2.2	79.0	102.7	73	1.1
Test item	40.0	+	107.0	95.3	207	2.0	84.9	146.5	37	0.6
Test item	80.0 (p)	+	82.4	67.6	251	2.4	67.6	134.9	45	0.7
Test item	160.0 (p)	+	88.6	51.2	237	2.2	75.7	52.6	80	1.2
Experiment II / 24 h treatment			culture I				culture II
Neg. control with medium		-	100.0	100.0	52		100.0	100.0	46
Solv. control with DMSO		-	100.0	100.0	34	1.0	100.0	100.0	37	1.0
POS. Control with MMS	13.0	-	35.7	29.2	756	14.6	42.8	19.1	392	8.5
Test item	1.3	-	116.4	culture was not continued*			114.4	culture was not continued*
Test item	2.5	-	93.7	culture was not continued*			102.5	culture was not continued*
Test item	5.0	-	100.0	59.0	48	1.4	87.5	67.6	51	1.4
Test item	10.0	-	95.7	48.7	69	2.0	91.3	72.8	38	1.1
Test item	20.0	-	86.4	30.3	62	1.8	79.4	59.4	35	1.0
Test item	30.0	-	40.6	27.7	65	1.9	43.4	24.2	65	1.8
Test item	40.0	-	5.8	9.0	45	1.3	9.9	9.4	38	1.0
Experiment II / 4 h treatment
Neg. control with medium		+	100.0	100.0	57		100.0	100.0	56
Solv. control with DMSO		+	100.0	100.0	64	1.0	100.0	100.0	52	1.0
Pos. control with CPA	4.5	+	19.1	33.5	385	6.7	20.3	26.7	284	5.5
Test item	5.0	+	82.4	culture was not continued*			124.8	culture was not continued*
Test item	10.0	+	100.0	141.7	32	0.5	77.0	106.6	60	1.2
Test item	20.0	+	72.0	111.2	52	0.8	74.4	98.2	54	1.0
Test item	40.0	+	73.2	89.4	51	0.8	87.8	62.2	79	1.5
Test item	80.0 (p)	+	73.2	72.6	42	0.7	97.7	93.3	48	0.9
Test item	160.0 (p)	+	36.3	37.4	46	0.7	87.8	42.8	49	0.9
Experiment III / 4 h treatment			culture I				culture II
Neg. control with medium		-	100.0	100.0	196		100.0	100.0	144
Solv. control with DMSO		-	100.0	100.0	144	1.0	100.0	100.0	154	1.0
Pos. Control with MMS	19.5	-	77.5	24.4	429	2.2	92.6	11.7	381	2.6
Test item	5.0	-	78.8	culture was not continued*			103.5	culture was not continued*
Test item	10.0	-	87.9	85.2	127	0.9	83.8	81.2	182	1.2
Test item	20.0	-	60.2	98.6	163	1.1	100.0	72.3	173	1.1
Test item	40.0	-	62.2	89.7	147	1.0	98.3	87.7	153	1.0
Test item	80.0 (p)	-	53.1	17.6	160	1.1	70.2	38.0	156	1.0
Test item	160.0 (p)	-	39.1	20.6	347	2.4	96.7	33.2	195	1.3

* culture was not continued since a minimum of four concentrations is required by the guidelines

(p) precipitation visible to the naked eye

Table 3. HISTORICAL DATA

Number of mutant colonies per 10^6 cells
4 h treatment
	Negative control		Positive control			Solvent control
	without S9 mix	with S9 mix	without S9 mix	with S9 mix		without S9 mix	with S9 mix
			MMS	DMNA	CPA
range:	41 - 208	41 -197	212 – 3321	203 - 847	209 – 1269	41 - 204	40 - 205
Mean value:	107	110	479	389	390	106	110
Standard deviation	37	38	281	203	170	39	35

Table 3. HISTORICAL DATA (cont.)

Number of mutant colonies per 10^6 cells
24 h treatment
	Negative control	Positive control	Solvent control
	without S9 mix	without S9 mix (MMS)	without S9 mix
range:	40-212	243 - 2431	40 - 202
Mean value:	109	863	107
Standard deviation	40	461	39

Conclusions:

The test material did not invoke a mutagenic response under the conditions of the test.

Executive summary:

In this OECD 476 test with GLP certification, mouse lymphoma cells L5178Y were treated with test article at doses up to 160 µg/mL using DMSO as a vehicle. The test design consisted of two main experiments using two parallel cultures.The cells were treated with the test item for 4 h with and without metabolic activation in the first experiment. In the second experiment the duration of treatment was 4 h with and 24 h without metabolic activation.A confirmatory experiment was added to verify mutagenicity data generated in the first experiment without metabolic activation. Experiment III was solely performed in the absence of metabolic activation under experimental conditions identical to the first experiment without metabolic activation.

Precipitation of the test item and decrease in cloning efficiency was observed at doses of 80 and 160 µg/mL. A biologically irrelevant increase in mutation frequency was observed in some dishes of the first experiment. They were not reproduced in the parallel culture and considered as artifacts caused by precipitates. An increase in mutation frequency was also observed in the second experiment (without metabolic activation, 24 h). Since absolute values of the mutation frequency remained within the historical range of negative and solvent controls, this effect was considered to be biologically irrelevant. Therefore, the test article is considered to be non-mutagenic to mammalian cells in vitro.

Reference 2

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:

1987-04-07, 1987-05-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1981

Deviations:

yes

Remarks:

Only four S. Typhimurium strains instead of five were used.

Principles of method if other than guideline:

The experiments were performed in a room with red light illumination, in order to avoid any influence of UV-light on the test material.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (All strains are histidine-auxotrophic mutants)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

0.3 mL S9 fraction of liver from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.7 mL of a solution of cofactors.

Test concentrations with justification for top dose:

20, 78, 313, 1250, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: test article is insoluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without activation

Positive control substance:

other: Daunorubicin-HCl

Remarks:

strain TA 98: 5 and 10 µg/plate

Positive controls:

yes

Remarks:

without activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

strain TA 100: 0.125 and 0.25 µg/plate

Positive controls:

yes

Remarks:

without activation

Positive control substance:

sodium azide

Remarks:

strain TA 1535: 2.5 and 5.0 µg/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without activation

Positive control substance:

9-aminoacridine

Remarks:

strain TA 1537: 50 and 100 µg/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

strains TA 98, TA 100, TA 1537: 5 µg/plate

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with activation

Positive control substance:

cyclophosphamide

Remarks:

strain TA 1535: 250 µg/plate

Details on test system and experimental conditions:

DETERMINATION OF CYTOTOXICITY
In a preliminary toxicity test with strain TA 100 without metabolic activation (plate incorporation), nine concentrations of test article ranging from 0.08 to 5000 µg/plate were tested to determine the highest concentration to be used in the mutagenicity assay.

MUTAGENICITY TEST
All experiments were performed in triplicate. The test was performed as agar plate incorporation test for 48 h in the dark at 37 +/- 1.5 °C. The selection based on growth in absence of histidine.

Evaluation criteria:

The test substance is considered to be positive when:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains: TA 98, TA 1535 and TA 1537;
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.

A test is considered acceptable if the mean colony counts of the control values of all strains are within the acceptable ranges and if the results of the positive controls meet the criteria for a positive response.

Statistics:

Arithmetic mean and standard deviation

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

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: At the concentrations of 313 µg/plate and above the test article precipitated in soft agar.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Table 1.

Table1. Toxicity test on strain TA 100 without microsomal activation. Colonies per plate and arithmetic mean (m).

Concentration μg/plate	Experiment
	1	2	mean
Control	113	113	113
0.08	104	109	107
0.31	115	138	127
1.2	114	120	117
4.9	108	121	115
19.5	111	124	118
78.1	104	115	110
312.5	114	124	125
1250.0	123	127	125
5000.0	113	116	115
Positive controls (4-nitroquinoline-N-oxide) (μg/mL)
Control	105	111	108
0.125	883	930	907
0.250	1387	1483	1435

Table 2. Mutagenicity test* without microsomal activation: number of back mutant colonies per plate, arithmentic mean (m) and standard deviation (SD)

Concentration μg/plate	STRAIN
	TA 98				TA 100				TA 1535				TA 1537
Experiment	1	2	3	mean (SD)	1	2	3	mean (SD)	1	2	3	mean (SD)	1	2	3	mean (SD)
Control	19	8	18	15 (6)	125	138	161	141 (18)	13	12	14	13 (1)	5	13	6	8 (4)
20	26	18	17	20 (5)	140	164	165	156 (14)	20	14	19	18 (3)	4	2	5	4 (2)
78	19	19	16	18 (2)	139	173	170	161 (19)	7	18	16	14 (6)	12	8	5	8 (4)
313	21	17	13	17 (4)	132	149	132	138 (10)	9	18	13	13 (5)	5	6	5	5 (1)
1250	11	15	14	13 (2)	152	124	153	143 (16)	14	9	15	13 (3)	3	4	4	4 (1)
5000	12	16	13	14 (2)	149	140	132	140 (9)	8	11	13	11 (3)	6	4	5	5 (1)
Positive controls (μg/plate)
	daunorubicin-HCl
Control	15	18	12	15 (3)
5	914	794	788	832 (71)
10	481	530	400	470 (66)
					4-nitroquinoline-N-oxide
Control					151	153	134	146 (10)
0.125					560	567	606	578 (25)
0.25					1144	892	1065	1034 (129)
									sodium azide
Control									15	14	5	11 (6)
2.5									484	448	524	485 (38)
5									836	776	750	787 (44)
													9 aminoacridine
Control													12	5	9	9 (4)
50													14	17	26	19 (6)
100													206	194	261	220 (36)

* representative study

Table 3. Mutagenicity test* with microsomal activation: number of back mutant colonies per plate, arithmentic mean (m) and standard deviation (SD)

Concentration μg/plate	STRAIN
	TA 98				TA 100				TA 1535				TA 1537
Experiment	1	2	3	mean (SD)	1	2	3	mean (SD)	1	2	3	mean (SD)	1	2	3	mean (SD)
Control	30	37	38	35 (4)	180	157	164	167 (12)	14	21	14	16 (4)	8	6	13	9 (4)
20	26	26	20	24 (3)	185	172	148	168 (19)	17	19	17	18 (1)	9	8	14	10 (3)
78	30	29	20	26 (6)	177	187	165	176 (11)	13	18	19	17 (3)	14	3	13	10 (6)
313	24	41	29	31 (9)	172	164	173	170 (5)	16	14	13	14 (2)	16	12	12	13 (2)
1250	28	24	25	26 (2)	197	192	171	187 (14)	9	11	12	11 (2)	6	9	8	8 (2)
5000	13	20	22	18 (5)	157	158	146	151 (7)	7	8	17	11 (6)	6	11	7	8 (3)
Positive controls (μg/plate)
	2-aminoanthracene				2-aminoanthracene								2-aminoanthracene
Control	36	17	25	26 (10)	161	163	148	157 (8)					8	12	8	9 (2)
5	1409	1287	762	1153 (344)	1779	1514	1416	1570 (188)					240	224	177	214 (33)
									cyclophosphamide
Control									6	17	12	12 (6)
250									409	325	414	383 (50)

* representative study

Conclusions:

The test material did not invoke a mutagenic response under the conditions of the test.

Executive summary:

In this study (OECD 471) conducted to generally accepted scientific standards with GLP, first a preliminary toxicity test with strain TA 100 (without metabolic activation) of the test material (EC 404-360-3) was conducted. Nine concentrations from 0.08 to 5000 µg/plate were tested without metabolic activation in order to determine the concentrations to be used for the main test.

For the main test, five concentrations ranging from 20 -5000 µg/plate (with a 0 µg/plate for control) were tested on different strains of Salmonella typhimurium with and without metabolic activation provided by rats' livers using acetone as a vehicle. Stastically, no mutagenic activity was observed over the duration of the test compared to the controls, thus according to the CLP regulation 1272/2008, this test material is not genotoxic.

Reference 3

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

Study period:

1989-11-07, 1990-02-26

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)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)

Version / remarks:

1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1984

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: Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units / mL / 100 µg / mL (Gibco AG, Basle, Switzerland).
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

0.15 mL S9 fraction of liver from rats (Tif: RAIf(SPF)) induced with Aroclor 1254 and 0.2 mL of a solution of cofactors in 0.65 mL medium.

Test concentrations with justification for top dose:

ORIGINAL STUDY
Experiment 1, 2, 3, 4, 5: 1.72, 3.44, 6.88, 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml

CONFIRMATORY STUDY
Experiment 1, 2: 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml
Experiment 3: 6.88, 13.75, 27.5, 55.0, 110.0 µg/ml
Experiment 4: 0.86, 1.72, 3.44, 6.88, 13.75 µg/ml

For further details see table 1.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The test substance is insoluble in water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: 0.1 and 0.04 µg/mL

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

0.15 mL S9 fraction of liver from RAI rats induced with Aroclor 1254 and 0.2 mL of a solution of cofactors and 0.65 ml medium. Migrated to IUCLID6: 40.0 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preincubation period of cells: 24 - 57 h

Exposure scenario in the original study:
Experiment 1: with activation, Duration of treatment: 3 hours, Recovery: 14 hours
Experiment 2: with activation, Duration of treatment: 3 hours, Recovery: 21 hours
Experiment 3: without activation, Duration of treatment: 14 hours, Recovery: 0 hours
Experiment 4: without activation, Duration of treatment: 24 hours, Recovery: 0 hours
Experiment 5: without activation. Duration of treatment: 48 hours, Recovery: 0 hours

Exposure scenario in the confirmatory study:
Experiment 1: with activation, Duration of treatment: 3 hours, Recovery: 14 hours
Experiment 2: with activation, Duration of treatment: 3 hours, Recovery: 21 hours
Experiment 3: without activation, Duration of treatment: 14 hours, Recovery: 0 hours
Experiment 4: without activation, Duration of treatment: 24 hours, Recovery: 0 hours

SPINDLE INHIBITOR (cytogenetic assays):
Colcemid 0.4 µg/mL

NUMBER OF METAPHASE EVALUATED:
200 in treatment groups, 50 in positive controls

NUMBER OF CELLS EVALUATED:
2000

DETERMINATION OF CYTOTOXICITY (performed as integral part of the mutagenicity test by mitotic index)
For the determination of the mitotic index the preparations from the various cultures were examined first, uncoded. The percentages of mitotic suppression in comparison with the controls were evaluated by counting at least 2000 cells per concentration.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes

Evaluation criteria:

200 metaphase figures with 19 to 21 chromosomes from cultures of two falcon flasks in the vehicle control and in the treated groups, and at least 50 metaphase cells in the positive controls were examined for the following aberrations:
a) specific aberrations: breaks, exchanges, deletions, fragments
b) unspecific aberrations: gaps, premature chromosome condensation, and chromosome decay
c) numerical aberrations (metaphases with >21 chromosomes) will be registered, yet reported only in case of deviations.

Statistics:

A test substance is considered to be active in this test system if in comparison to the negative control a marked increase in the number of specific chromosomal aberrations appears or if an increased number of exchange figures appear together with a high number of other specific chromosomal aberrations such as breaks and fragments.
A concentration-related response in the number of aberrations should be demonstrable.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Chromosome aberration and polyploidy

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index decrease (table 2).

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

REMARK
In the original study, in all experiments performed, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. The incidence of changes observed is within the range of spontaneous aberrations inherent in this particular cell line used as compared to the historical data. The confirmatory study confirmed the results of the original study.

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: TK 11319 is insoluble
- Precipitation: Addition of the stock solutions to the cell cultures produced turbidity in the media at the final concentrations of 220.0 and 110.0 µg/mL without and with activation.

Remarks on result:

other: strain/cell type: CHO-K1 CCL61

Remarks:

Migrated from field 'Test system'.

Table 2. Mitotic Index values (MI) Original study

Experiment 1. 3 hours with metabolic activation (recovery 14 h)
		Cells scored	Mitosis	Frequency % of control	MI (%)
Solvent control		2000	155	100	7.75
Test compound (μg/mL)	220.0	2000	54	34.8	2.7
	110.0	2000	65	41.9	3.25
	55.0	2000	78	50.3	3.9
	27.5	2000	117	75.5	5.85
	13.8	2000	145	93.5	7.25
	6.9	2000	150	96.8	7.5
	3.4	*
	1.7	*
Experiment 2. 3 hours with metabolic activation (recovery 21 h)
		Cells scored	Mitosis	Frequency % of control	M.I. (%)
Solvent control		2000	174	100	8.7
Test compound (μg/mL)	220.0	2000	71	40.8	3.55
	110.0	2000	99	56.9	4.95
	55.0	2000	116	66.7	5.8
	27.5	2000	145	83.3	7.25
	13.8	2000	135	77.6	6.75
	6.9	2000	152	87.4	7.6
	3.4	*
	1.7	*
Experiment 3. 14 hours without metabolic activation
		Cells scored	Mitosis	Frequency % of control	M.I. (%)
Solvent control		2000	135	100	6.75
Test compound (μg/mL)	220.0	2000	20	14.8	1.0
	110.0	2000	35	25.9	1.75
	55.0	2000	35	25.9	1.75
	27.5	2000	42	31.1	2.1
	13.8	2000	58	43	2.9
	6.9	2000	87	64.4	4.35
	3.4	2000	120	88.9	6.0
	1.7	2000	136	100.7	6.8
Experiment 4. 24 hours without metabolic activation
		Cells scored	Mitosis	Frequency % of control	M.I. (%)
Solvent control		2000	91	100	4.55
Test compound (μg/mL)	220.0	2000	1	1.1	0.05
	110.0	2000	18	19.8	0.9
	55.0	2000	18	19.8	0.9
	27.5	2000	16	17.6	0.8
	13.8	2000	66	72.5	3.3
	6.9	2000	73	80.2	3.65
	3.4	2000	75	82.4	3.75
	1.7	*
Experiment 5. 48 hours without metabolic activation
		Cells scored	Mitosis	Frequency % of control	M.I. (%)
Solvent control		2000	79	100	3.95
Test compound (μg/mL)	220.0	2000	4	5.1	0.2
	110.0	2000	10	12.7	0.5
	55.0	2000	7	8.9	0.35
	27.5	2000	10	12.7	0.5
	13.8	2000	19	24.1	0.95
	6.9	2000	64	81	3.2
	3.4	2000	82	103.8	4.1
	1.7	2000	93	117.7	4.65

* When 3 subsequent concentrations with a frequency of 70 % mitosis or more in relation to the negative control are found, the evaluation of the lower concentrations is omitted.

Conclusions:

The test material did not invoke a mutagenic response under the conditions of the test.

Executive summary:

In this OECD 473 test with GLP compliance, chinese hamster ovary cells CCL61 were treated with test article at doses up to 220 µg/ml using DMSO as a vehicle. The original test design consisted of five main experiments using two parallel cultures. The cells were treated with the test item for 3 h with metabolic activation and a recovery time of 14 hours and 21 hours (experiments 1 and 2 respectively). For experiments 3 -5, the treatment was with no metabolic activation or recovery time, and the durations of the treatments were 14 hours, 24 hours and 48 hours respectively.

A confirmatory experiment was added to verify data generated in the first set of experiments at doses up to 220 µg/ml

. These additional experiments were performed with metabolic activation with a treatment duration of three hours (experiments 1 and 2) and a recovery period of 14 and 21 hours (experiments 1 and 2 respectively). Experiments were also performed without metabolic activation with a treatment duration of 14 and 24 hours (experiments 3 and 4 respectively) without a recovery period.

In the original study, in all experiments performed, the number of cells with specific chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. The incidence of changes observed is within the range of spontaneous aberrations inherent in this particular cell line used as compared to the historical data. The confirmatory study confirmed the results of the original study. Thus according to this study, EC 404 -360 -3 is not genotoxic.

Genetic toxicity in vivo

Description of key information

Study conducted to recognised training guidelines with GLP

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-04-06, 1987-09-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

hamster, Chinese

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln, Switzerland
- Age at study initiation: Females 6 - 10 weeks; males 4 - 9 weeks
- Weight at study initiation: Females 23-32 g; males 26-35 g
- Assigned to test groups randomly: yes
- Diet: Ad libitum, standard diet: NAFAG No.924
- Water: Ad libitum
- Acclimation period: 4 - 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 51 - 74
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Carboxymethylcellulose (CMC) 0.5 % in water
- Justification for choice of solvent/vehicle: TK 11319 is insoluble in water
- Concentration of test material in vehicle: 5000 mg/kg bw in 20 mL/kg bw

Duration of treatment / exposure:

Single dose exposure

Frequency of treatment:

Single dose

Post exposure period:

16, 24, and 48 hours

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

In a preliminary toxicity test the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay.

No. of animals per sex per dose:

8 per sex per group

Control animals:

yes, concurrent no treatment

Positive control(s):

A dose of 64 mg/kg bw of cyclophosphamide was used as positive control. The route of administration was oral per gavage.

Tissues and cell types examined:

Bone marrow was harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture were transferred on the end of a slide, spread out with the aid of a polished cover glass. The preparations were air-dried. Within 24 hours, the slides were stained in undiluted May-Grünwald solution for 3 min then in May-Grünwald solution/water (1/1) for 2 min. After being rinsed in distilled water, the slides were left immersed in diluted Giemsa solution (16.6 %) for 10 min. After rinsing with distilled water and air-drying, the slides were cleared in xylene and mounted. 1000 polychromatic erythrocytes (PCE) per animal were used to determine the incidence of micronucleated PCE. A total of 1000 erythrocytes were counted for determination of the ratio of polychromatic to normochromatic erythrocytes (PCE/NCE -ratio).

Statistics:

The significance of difference is assessed by χ2-test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No significant differences in the PCE/NCE-ratio between treated groups and controls.

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1. Effect of test article on bone marrow cells of chinese hamster: arithmetic mean per sex and group.

	Sacrifice (h)
	16				24				48
	Control		TK 11319		Control		TK 11319		Control		TK 11319
sex of the animals	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂	♀	♂
polychromatic erythrocytes (P)	626	520	550	551	489	471	482	486	555	539	459	485
normochromatic erythrocytes (N)	374	480	450	449	511	529	518	514	445	461	541	515
ratio P/N	1.7	1.1	1.2	1.2	1.0	0.9	0.9	0.9	1.2	1.2	0.8	0.9
P with micronuclei	0.2	0.2	0.4	0.2	0.6	1.0	1.0	0.6	0.8	0.8	0.6	0.6
percent of P with micronuclei	0.02	0.02	0.04	0.02	0.06	0.10	0.10	0.06	0.08	0.08	0.06	0.06

Table 1 (cont.). Effect of test article on bone marrow cells of chinese hamster: arithmetic mean per sex and group (positive control).

	Sacrifice (h)
	24
	Control		Cyclophosphamide (64 mg/kg)
sex of the animals	♀	♂	♀	♂
polychromatic erythrocytes (P)	489	471	452	450
normochromatic erythrocytes (N)	511	529	548	550
ratio P/N	1.0	0.9	0.8	0.8
P with micronuclei	0.6	1.0	27.0	32.2
percent of P with micronuclei	0.06	0.10	2.70	3.22

Conclusions:

The substance tested in the in vivo Micronucleus Test with Chinese Hamster (method similar to OECD 474) up to 5000 mg/kg bw was not genotoxic.

Executive summary:

In this guideline (OECD 474) study conducted to GLP standards, the test material (EC 404-360-3) was tested in vivo on chinese hamsters (male and female) aged 4 -10 weeks. The test substance was administered via oral gavage in a vehicle of 0.5% carboxymethylcellulose (concentration of test material in vehicle was 5000 mg/kg bw in 20 ml/kg bw). 8 animals of each sex per dose were tested, with various sacrifice times (16, 24 and 48 hours). After sacrifice, bone marrow was harvested from the shafts of both femurs with fetal calf serum for analysis. As well as a control, there was a positive control using 64 mg/kg bw of cyclophosphamide (also administered via oral gavage). The bone marrow tissue was then examined for incidence of polychromatic erythrocytes (PCE) - an increase in frequency being an indicator of induced chromosome damage. No PCE was observed for the test material, therefore EC 404 -360 -3 is not genotoxic.

Additional information

Two Ames tests are available. Concentrations of 20, 78, 313, 1250, and 5000 µg/plate were tested in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with and without metabolic activation (Ciba-Geigy 1987) according to OECD 471 (1981). The same concentrations were tested in a previous screening study in Salmonella strains TA 98, TA 100 and TA 1537, with and without metabolic activation (Ciba-Geigy 1986); the test conduct was similar to OECD 471.

In the first study no increased mutation frequency was observed both in the absence and presence of S9-mix. Up to the highest concentration no cytotoxicity was seen. In the second study also no increased mutation frequency was observed in the absence of S9-mix. In the presence of S9-mix, a slight (not doubled) and not significant increase of mutant colonies was observed at concentrations of 313 µg/plate and above for strain TA 100 only. Since the mutation frequency was not doubled in comparison to control and no dose-relationship was seen the effect is considered of no biological relevance. Up to the highest concentration no cytotoxicity was seen.

Thus, the test article was not genotoxic when tested in the Ames test under the chosen conditions.

In a guideline study conducted according to OECD 473 (1983) doses of 1.72, 3.44, 6.88, 13.75, 27.5, 55.0, 110.0, 220.0 µg/ml were tested for the induction of chromosome aberrations in Chinese hamster ovary cells in vitro with and without S9-mix (Ciba-Geigy 1990). The number of cells with chromosomal aberrations in the treatment groups showed no marked increase in comparison with the negative control. Thus, under the given experimental conditions, no evidence of mutagenic effects was obtained in Chinese hamster ovary cells in vitro after treatment with the test article.

Mouse lymphoma cells L5178Y were treated with test article according to OECD 476 (RCC 2006). The test design consisted of two main experiments using two parallel cultures. The cells were treated with the test item for 4 h with and without metabolic activation in the first experiment. In the second experiment the duration of treatment was 4 h with and 24 h without metabolic activation. A confirmatory experiment was added to verify mutagenicity data generated in the first experiment without metabolic activation. The cultures were evaluated at concentrations ranging from 10 to 160 µg/ml with and without S9-mix in the first experiment. In the second experiment the concentrations were 5 to 40 µg/ml without and 10 to 160 µg/ml with S9-mix. The confirmatory experiment without S9-mix was conducted with 10 to 160 µg/ml.

Precipitation of the test item and decrease in cloning efficiency was observed at doses of 80 and 160 µg/ml in all experiments. A biologically irrelevant increase in mutation frequency was observed in some dishes of the first experiment with S9-mix, which could not be reproduced; the effect was therefore considered as artifact. An increase in mutation frequency was also observed in the second experiment (without S9-mix). Since absolute values of the mutation frequency remained within the historical range of negative and solvent controls, this effect was also considered to be biologically irrelevant. Therefore, the test article was not mutagenic in the mouse lymphoma test.

An in vivo micronucleus test with Chinese Hamster was conducted similar to OECD 474, at a dose of 5000 mg/kg bw (Ciba-Geigy 1987).The number of polychromatic erythrocytes with micronuclei in the treatment group showed no increase in comparison to the negative control. Thus, under the given experimental conditions, no evidence of mutagenic effects was obtained in Chinese hamster in vivo.

Under in vitro and in vivo conditions, the substance is not mutagenic to bacteria and to mammalian cells in culture with and without metabolic activation and is not clastogenic in mammalian cells. Under in vivo conditions the substance is not clastogenic in Chinese hamster.

 

Short description of key information:
Valid studies on the in vitro and in vivo genetic toxicity are available. Two Ames tests were performed, the more recent according to OECD 471 (Ciba-Geigy 1987) and the older as a screening study with three strains (Ciba-Geigy 1986). A chromosome aberration assay with Chinese Hamster Ovary Cells was conducted according to OECD 473 (Ciba-Geigy 1990) and a forward mutation assay in mouse lymphoma L5178Y cells according to OECD 476 (RCC, 2006) is also available. For in vivo mutagenicity testing, a micronucleus test with Chinese Hamster similar to OECD 474 was done (Ciba-Geigy1987). None of the assays revealed positive results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for mutagenicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

 

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 is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive (EC 944/2013).