2-{[4-({4-[(4-amino-6-chloro-1,3,5-triazin-2-yl)amino]-5-sulfo-1-naphthyl}diazenyl)-(6 or 7)-sulfo-1-naphthyl]diazenyl}benzene-1,4-disulfonic acid, lithium sodium salts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation(OECD TG471).

Read-across from supporting substance, CJ313 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation (OECD TG473).

CJ313 was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay(OECD TG476).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 17, 2017 to July 03, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) prepared from uninduced male Golden Syrian Hamster liver

Untreated negative controls:

yes

Remarks:

Sterile deionized water

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

congo red

mitomycin C

other: Acridine mutagen ICR 191, 2-Aminofluorene, 2-Amoinoanthracene

Evaluation criteria:

Tester Strain Revertants
TA98 10-60
TA100 50-240
TA102 180-480
TA1535 5-45
TA1537 2-25

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 μg/plate

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The substance was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 ug/plate.

Table 1. Genotype Confirmation Test of Salmonella typhimurium Tester Strains

Genotype character	Phenotypic observation	Tester Strains
		TA98	TA100	TA102	TA1535	TA1537
Histidine requirement	growing on biotin plate	－	－	－	－	－
	growing on histidine/biotin plate	+	+	+	+	+
rfamutation	inhibition zone of crystal violet	+	+	+	+	+
ΔuvrBmutation	growing on non UV-irradiated plate	+	+	+	+	+
	growing on UV-irradiated plate	－	－	+	－	－
R-factor	ampicillin resistance	+	+	+	－	－
Genotype confirmed		Passed	Passed	Passed	Passed	Passed

+: the presence

－: the absence

 

Table 2. Mutagenicity Test of CJ313 in Salmonella typhimurium Strains without S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
Replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	27	25	31	50	53	70	288	322	268	16	6	13	21	14	15
	M^f	28 ± 3			58 ± 11			293 ± 27			12 ± 5			17 ± 4
50	I^e	29	23	30	56	61	53	322	336	318	18	12	16	4^h	9	12
	M^f	27 ± 4			57 ± 4			325 ± 9			15 ± 3			8^g± 4
150	I^e	14	18	18	58	71	52	216	350	280	13	16	22	17	11	14
	M^f	17 ± 2			60 ± 10			282 ± 67			17 ± 5			14 ± 3
500	I^e	18	17	26	57	49	80	332	256	204	12	15	16	6	12	16
	M^f	20 ± 5			62 ± 16			261 ± 59			14 ± 2			11 ± 5
1500	I^e	23	25	23	70	61	58	294	294	278	14	17	16	17	10	9
	M^f	24 ± 1			63 ± 6			289 ± 9			16 ± 2			12 ± 4
5000	I^e	25	22	17	42	60	57	258	176	292	13	11	18	10	13	16
	M^f	21 ± 4			53 ± 10			242 ± 60			14 ± 4			13 ± 3
Positive control^b	I^e	181	255	266	374	374	430	1411	1229	1376	439	428	349	191	181	174
	M^f	234^c± 46			393^c±32			1339^c± 97			405^d± 49			182^d± 9

a: Negative control was sterile deionized water.

b: Positive controls: 1 μg/plate 2-nitrofluorene for TA98

0.5 μg/plate sodium azide for TA100

62.5 ng/plate mitomycin C for TA102

0.1 μg/plate sodium azide for TA1535

0.3 μg/plate acridine mutagen ICR 191 for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

h: Extreme low data may consider as an outlier (out of range of historical data).

 

Table 3. Mutagenicity Test of CJ313 in Salmonella typhimurium Strains with S9 Metabolic Activation

Treatment (μg/plate)		Number of Revertant Colonies in Salmonella typhimurium
		TA98			TA100			TA102			TA1535			TA1537
Replicate		1	2	3	1	2	3	1	2	3	1	2	3	1	2	3
Negative control^a	I^e	38	41	38	86	75	89	515	486	562	15	11	13	29	33	32
	M^f	39 ± 2			83 ± 7			521 ± 38			13 ± 2			31 ± 2
50	I^e	28	40	37	63	108	70	337	414	483	12	14	18	28	22	19
	M^f	35 ± 6			80 ± 24			411 ± 73			15 ± 3			23 ± 5
150	I^e	43	38	47	61	76	88	422	416	131	7	15	11	26	24	31
	M^f	43 ± 5			75 ± 14			384 ± 61			11 ± 4			27 ± 4
500	I^e	23	36	41	84	88	67	296	316	390	15	16	17	29	35	34
	M^f	33 ± 9			80 ± 11			334 ± 50			16 ± 1			33 ± 3
1500	I^e	36	35	33	93	77	90	294	367	391	12	12	11	18	18	13
	M^f	35 ± 2			87 ± 9			351 ± 51			12 ± 1			16 ± 3
5000	I^e	27	27	17	65	54	80	343	179	136	10	11	7	10	8	16
	M^f	24 ± 6			66 ± 13			219^g± 109			9 ± 2			11^g± 4
Positive control^b	I^e	263	329	312	254	298	346	1133	1336	1086	300	307	459	146	132	110
	M^f	301^c± 34			299^c± 46			1185^c± 133			355^d± 90			129^d± 18

a: Negative control was sterile deionized water.

b: Positive controls: 60 μg/plate Congo Red for TA98

1 μg/plate 2-aminofluorene for TA100

2 μg/plate 2-aminoanthracene for TA102

0.5 μg/plate 2-aminoanthracene for TA1535

2 μg/plate 2-aminoanthracene for TA1537

c: Greater than 2-fold negative control spontaneous revertants

d: Greater than 3-fold negative control spontaneous revertants

e: I: Number of revertants/plate is shown for each individual plate.

f: M: The value of mean ± S.D. from triplicate plates of each treatment was calculated.

g: Cytotoxicty: a > 50% reduction when compared to the mean colony number of negative control revertants.

Conclusions:

According to OECD 471 test method, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316020-GT which is based on the SOP for the OECD 471 (CTPS-TE00201) and OECD 471 (OECD, 1997). The results of this OECD 471 test for CJ313 show that test validity criteria was met.

Based on the preliminary assay results, 5000 μg/platewas set as the highest dose in this study. In the mutagenicity assay, five doses of CJ313 at 50, 150, 500, 1500 and 5000 μg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From January 11, 2017 to June 07, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The read-across target has structure similarity to CJ313. Therefore, the data is applied.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1

Metabolic activation:

with and without

Metabolic activation system:

The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague- Dawley rats

Untreated negative controls:

yes

Remarks:

Sterile deionized water

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1.Karyology Analysis of Chinese Hamster Ovary Cells

No. of chromosome	<18	18	19	20	21	22	>22
No. of cells	0	0	9	35	5	1	0

Table 2. Concurrent Cytotoxicity Analysis of CJ309 in Chinese Hamster Ovary Cells

Concentration (μg/mL)	Cell Number (× 10^5cells)	ICC^a N-N0	RICC^b (%)	Cytotoxicity^c (%)
Before Treatment
Untreated (-S9)	30.4
Untreated (+S9)	26.4
After Treatment
Scheme I (-S9, 3h)
Negative Control	63.6	33.2	100.0	0.0
51.2	62.2	31.8	95.8	4.2
128	67.0	36.6	110.2	0.0
320	65.0	34.6	104.2	0.0
800	62.6	32.2	97.0	3.0
2000	61.2	30.8	92.8	7.2
Positive Control^d	48.6	18.2	54.8	45.2
Scheme II (+S9, 3h)
Negative Control	55.8	29.4	100.0	0.0
51.2	54.4	28.0	95.2	4.8
128	54.6	28.2	95.9	4.1
320	55.6	29.2	99.3	0.7
800	49.0	22.6	76.9	23.1
2000	52.8	26.4	89.8	10.2
Positive Control^e	44.6	18.2	61.9	38.1
Scheme III (-S9, 20h)
Negative Control	63.8	33.4	100.0	0.0
51.2	57.6	27.2	81.4	18.6
128	110.2	79.8	238.9	0.0
320	108.0	77.6	232.3	0.0
800	54.4	24.0	71.9	28.1
2000	58.8	28.4	85.0	15.0
Positive Control^f	56.8	26.4	79.0	21.0

a ICC: increased in cell counts = Cell No._{After treatment}(N) - Cell No._{Before treatment}(N₀)

b RICC: relative increase in cell counts; RICC = (ICC_treatment/ICC_control) × 100

c Cytotoxicity (%) = 100 – RICC

d Positive control was 0.33μg/mL mitomycin C (MMC)

e Positive control was 11.2μg/mL cyclophosphamide (CPP)

f Positive control was 0.2μg/mL mitomycin C (MMC)

 

Table 3. Summary of Chromosome Aberrations in Chinese Hamster Ovary Cells for CJ309

Treatment	Concentration (μg/mL)	Treating Hour	S9 (-/+)	Aberrant Cells (%)
Scheme I (-S9, 3h)
Negative Control^a	0	3	-	0.33
Test Article	51.2	3	-	0.33
	128	3	-	0.67
	320	3	-	0.33
	800	3	-	0
	2000	3	-	1
Positive Control^c	0.33	3	-	19.33*
Scheme II (+S9, 3h)
Negative Control^b	0	3	+	0
Test Article	51.2	3	+	0.67
	128	3	+	0.67
	320	3	+	0.67
	800	3	+	1
	2000	3	+	0.33
Positive Control^d	11.2	3	+	24*
Scheme III (-S9, 20h)
Negative Control^a	0	20	-	0.33
Test Article	51.2	20	-	0.33
	128	20	-	0.67
	320	20	-	1
	800	20	-	1.33
	2000	20	-	−
Positive Control^e	0.2	20	-	29*

All data were scored from 300 metaphase cells of each treatment (duplicate cultures).

a: Negative control was 10% sterile deionized water in McCoy’s 5A medium.

b: Negative control was 10% sterile deionized water in S-9 mixture medium.

c: Positive control was 0.33μg/mL mitomycin C (MMC).

d: Positive control was 11.2μg/mL cyclophosphamide (CPP).

e: Positive control was 0.2μg/mL mitomycin C (MMC).

*: The frequency of aberrant cells was significantly higher than that of the negative control (One-tailed binomial test, α = 0.01).

−: Too few metaphases

Conclusions:

According to OECD 473 test method, CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65316027-GT and OECD 473 (OECD, 2016). The results of this OECD 473 test for CJ309 show that test validity criteria was met.

A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition,CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From March 31, 2016 to May 26, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The main structure of the read-across substance is the same of the CJ313, only the salt form is different. However, this difference does not influence the toxicity of the CJ313. CJ313 contains Li/Na salt form and the read-across substances contains only Na salt. This will not lead to difference on the toxicity results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation tests using the Hprt and xprt genes

Specific details on test material used for the study:

Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light. Dose formulations were adjusted to compensate for the purity (84.3%) of the test substance, using a correction factor of 1.19.

Target gene:

The purpose of this study is to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) in Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6 thioguanine (TG resistance, TGr)

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- 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:

Arcolor 1254-induced rat liver S9

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
In the definitive mutagenicity assay, the concentrations tested were 125, 250, 500, 1000 and 2000 µg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 µg/mL without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water (test article); DMSO (Ethylmethanesulfonate, Benzo(a)pyrene)
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assay

NUMBER OF CELLS EVALUATED: 2.4 x 10E6 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival

Rationale for test conditions:

None

Evaluation criteria:

The test substance was considered to have produced a positive response if it induced a dose-related increase in mutant frequency and an increase exceeding the 95 % historical vehicle control limits in at least one test dose level as compared with the concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.

Statistics:

Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Preliminary toxicity assay: The pH of the cultures was adjusted at all concentrations with and without S9 to maintain neutral pH; Definitive mutagenicity assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.29 at the top dose).
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120 % in the preliminary toxicity assay.
- Precipitation: No visible precipitate was observed at the beginning or end of treatment in both the assays.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1. Summary Results for FAT 40032/G Preliminary Toxicity Assay ±S9

Treatment	Dose Level	S9	Initial Survival
			Cells/mL	Cloning Efficiency				Relative Survival
	(µg/mL)		(x 10^6)	(Colonies/Plate)			(%)	(adj., %)
di-H2O	100.0^a	-	0.230	145	150	140	72.50	100.00
FAT 40032/G	3.91	-	0.217	105	103	128	56.00	72.99
FAT 40032/G	7.81	-	0.230	160	135	156	75.17	104.08
FAT 40032/G	15.6	-	0.231	151	169	151	78.50	108.95
FAT 40032/G	31.3	-	0.240	215	192	197	100.67	145.04
FAT 40032/G	62.5	-	0.225	143	145	135	70.50	95.30
FAT 40032/G	125	-	0.223	162	170	169	83.50	111.64
FAT 40032/G	250	-	0.206	145	181	143	78.17	96.86
FAT 40032/G	500	-	0.204	127	122	128	62.83	76.96
FAT 40032/G	1000	-	0.116	146	168	140	75.67	52.73
FAT 40032/G	2000	-	0.023	29	26	38	15.50	2.12
di-H2O	100.0^a	+	0.196	150	150	160	76.67	100.00
FAT 40032/G	3.91	+	0.223	107	113	134	59.00	87.82
FAT 40032/G	7.81	+	0.219	168	183	162	85.50	124.84
FAT 40032/G	15.6	+	0.232	137	137	126	66.67	103.08
FAT 40032/G	31.3	+	0.222	171	135	169	79.17	117.13
FAT 40032/G	62.5	+	0.216	154	147	146	74.50	107.43
FAT 40032/G	125	+	0.228	169	144	159	78.67	119.46
FAT 40032/G	250	+	0.245	125	95	120	56.67	92.49
FAT 40032/G	500	+	0.259	144	166	166	79.33	137.23
FAT 40032/G	1000	+	0.225	144	144	140	71.33	107.08
FAT 40032/G	2000	+	0.173	158	158	185	83.50	96.59

^a (µL/mL)

Table 2. Summary Results for FAT 40032/G Definitive Mutagenicity Assay -S9

Treatment	Dose Level	S9	Initial Survival						TGr Mutants/Plate				Total Mutant Colonies	Selection
			Cells/mL	Cloning Efficiency				Relative Survival						Cloning Efficiency				Mutant Frequency (x 10^-6)
	(µg/mL)		(x 10^6)	(Colonies/Plate)			(%)	(adj., %)						(Colonies/Plate)			(%)	Individual	Average
di-H2O	100.0^a	-	0.738	172	172	172	86.00	95.82	4	4	3	3	14	172	180	187	89.83	6.49	5.88
di-H2O	100.0^a	-	0.923	186	179	196	93.50	104.18	3	5	3	2	13	211	203	203	102.83	5.27
FAT 40032/G	125	-	0.977	151	140	159	75.00	83.57	3	10	4	2	19	167	185	138	81.67	9.69	7.20
FAT 40032/G	125	-	0.948	138	126	151	69.17	77.07	1	3	1	4	9	167	167	144	79.67	4.71
FAT 40032/G	250	-	0.644	189	147	162	83.00	92.48	3	3	1	0	7	151	194	155	83.33	3.50	6.48
FAT 40032/G	250	-	0.934	144	153	186	80.50	89.69	7	3	8	1	19	182	150	170	83.67	9.46
FAT 40032/G	500	-	0.457	173	179	177	88.17	98.24	2	1	1	3	7	148	138	156	73.67	3.96	4.79
FAT 40032/G	500	-	0.554	158	160	156	79.00	88.02	3	3	2	4	12	179	196	158	88.83	5.63
FAT 40032/G	1000	-	0.262	164	176	144	80.67	89.88	7	2	4	5	18	160	170	192	87.00	8.62	10.17
FAT 40032/G	1000	-	0.492	180	159	185	87.33	97.31	5	4	9	C	18	169	172	171	85.33	11.72
FAT 40032/G	1200	-	0.338	179	147	147	78.83	87.84	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	1200	-	0.284	203	171	197	95.17	106.04	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	1500	-	0.187	163	172	150	80.83	90.06	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	1500	-	0.131	146	151	159	76.00	84.68	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	1800	-	0.182	201	186	191	96.33	107.34	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	1800	-	0.138	170	179	160	84.83	94.52	ND	ND	ND	ND	ND	ND	ND	ND	ND	ND
FAT 40032/G	2000	-	0.144	160	159	171	81.67	90.99	6	4	9	4	23	218	208	235	110.17	8.7	6.90
FAT 40032/G	2000	-	0.165	193	165	179	89.50	99.72	2	1	4	4	11	177	187	176	90	5.09
EMS	0.200^a	-	1.046	111	117	100	54.67	60.91	104	84	79	86	353	130	162	148	73.33	200.57	250.55**
EMS	0.200^a	-	1.091	123	103	112	56.33	62.77	119	108	112	119	458	122	131	128	63.50	300.52

^a (µL/mL)

C = Contaminated

ND = Not determined

** p <=0.01

Table 3. Summary Results for FAT 40032/G Definitive Mutagenicity Assay +S9

Treatment	Dose Level	S9	Initial Survival						TGr Mutants/Plate				Total Mutant Colonies	Selection
			Cells/mL	Cloning Efficiency				Relative Survival						Cloning Efficiency				Mutant Frequency (x 10^-6)
	(µg/mL)		(x 10^6)	(Colonies/Plate)			(%)	(adj., %)						(Colonies/Plate)			(%)	Individual	Average
di-H2O	100.0^a	+	0.647	156	139	146	73.50	97.24	3	4	3	8	18	151	136	152	73.17	10.25	12.29
di-H2O	100.0^a	+	0.707	151	170	145	77.67	102.76	4	14	7	7	32	159	191	208	93	14.34
FAT 40032/G	125	+	0.748	190	177	152	86.50	114.44	2	2	2	3	9	146	139	136	70.17	5.34	4.37
FAT 40032/G	125	+	0.763	158	142	160	76.67	101.43	2	3	0	1	6	150	143	149	73.67	3.39
FAT 40032/G	250	+	0.767	157	168	137	77.00	101.87	2	6	3	7	18	178	139	152	78.17	9.59	9.97
FAT 40032/G	250	+	0.730	139	163	176	79.67	105.40	12	4	2	2	20	172	159	152	80.5	10.35
FAT 40032/G	500	+	0.773	165	179	171	85.83	113.56	8	4	9	9	30	146	192	157	82.5	15.15	11.25
FAT 40032/G	500	+	0.727	153	160	153	77.67	105.76	2	4	3	6	15	167	170	173	85	7.35
FAT 40032/G	1000	+	0.689	143	152	152	74.50	98.57	5	4	2	2	13	184	201	170	92.50	5.86	6.53
FAT 40032/G	1000	+	0.706	142	169	166	79.50	105.18	3	4	4	4	15	170	177	C	86.75	7.2
FAT 40032/G	2000	+	0.501	155	130	145	71.67	94.82	2	8	2	7	19	164	165	165	82.33	9.62	7.83
FAT 40032/G	2000	+	0.428	143	132	118	65.50	86.66	8	2	2	0	12	169	151	176	82.67	6.05
B(a)P	4.000	+	0.702	103	74	96	45.50	60.20	59	52	57	44	212	123	127	165	69.17	127.71	133.39**
B(a)P	4.000	+	0.740	86	86	92	44.00	58.21	64	55	57	71	247	159	137	148	74.00	139.08

^a (µL/mL)

C = Contaminated

** p <=0.01

Conclusions:

Under the conditions of the assay described in this report, FAT 40032/G was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.

Executive summary:

The test substance, FAT 40032/G, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Water was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 μg/mL without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 μg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). All control data were within the 95% control limits except for the solvent control mutant frequency data with S9. However, these values were acceptable as they were within the observed historical control range. The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate FAT 40032/G was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro gene mutation study in bacteria

Based on the preliminary assay results, 5000 μg/plate was set as the highest dose in this study. In the mutagenicity assay, five doses of CJ313 at 50, 150, 500, 1500 and 5000 μg/plate, concurrent negative and strain-specific positive controls were tested in tester strains TA98, TA100, TA102, TA1535 and TA1537 in triplicate with or without S9 Mix activation.Cytotoxicity was observed in tester strains TA102 and TA1537 at 5000 μg/plate in the presence of metabolite activation. Unexpected greater than 50% reduction of negative control revertants was observed in tester strain TA1537 at 50 μg/plate but would not consider cytotoxicity in the absence of metabolite activation. Results showed that CJ313 did not increase the number of revertants in all five tester strains TA98, TA100, TA102, TA1535 and TA1537 in the absence of metabolite activation, three tester strains TA98, TA100 and TA1535 in the presence of metabolite activation up to 5000 μg/plate; two tester strains TA102 and TA1537 in the presence of metabolite activation up to 1500 μg/plate. Based on the data obtained from this study, it was concluded that under the test condition, CJ313 was not mutagenic in the reverse mutation analysis of Salmonella typhimurium up to 1500 μg/plate in the absence and presence of S9 metabolic activation.

in vitro cytogenicity / chromosome aberration study in mammalian cells

A preliminary concentration-range finding cytotoxicity test was performed in three test schemes: 3-hour treatment in the absence and presence of S9 metabolic activation (Schemes I and II) and 20-hour continuous treatment in the absence of S9 metabolic activation (Scheme III), and the results suggested that a concentration of 2000μg/mL in all three schemes would be used in the chromosome aberration assay. Based on the cytotoxicity result, five concentrations of 51.2, 128, 320, 800 and 2000μg/mL were used for all three schemes in the chromosome aberration test. In test group, the cell proportions with abnormal chromosome in three Schemes were not higher than 3%. Based on the data obtained from this study, it was concluded that under the test condition,CJ309 did not induce chromosome aberration in CHO cells in absence or presence of S9 metabolic activation.

in vitro gene mutation study in mammalian cells

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 μg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12% at a concentration of 2000 μg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 μg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 μg/mL without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36% at a concentration of 2000 μg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 μg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). All control data were within the 95% control limits except for the solvent control mutant frequency data with S9. However, these values were acceptable as they were within the observed historical control range. The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate FAT 40032/G was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.