Sodium 4-(4-substitutedanilino)-4-substituedbut-2-enoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Apr - 21 Apr 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

(duplicate instead of triplicate plating was performed)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

duplicate instead of triplicate plating was performed

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: SystemsGuidelines for Japanese Industrial Safety and Health Act and Chemical Substance Control Law

Version / remarks:

Criteria for Mutagenicity Test in Bacterial Systems (Ministry of Labour, Japan, Notification No.77 dated September 1, 1988 and Notification No.67 dated J une 2, 1997)

Japanese Ministry of Health, Labor and Welfare, Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of the Environment, Heisei 23.3.31 Yakushokuhatsu Notification No. 0331-7, Heisei 23.3.29 Seikyoku Notification No. 5, Kanpokihatsu Notification No. 110331009

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the solvent/vehicle: ≥ 50 mg/mL in water, stable for 20 hours at room temperature and prepared just before use

FORM AS APPLIED IN THE TEST: liquid

Method

Target gene:

his operon, tryp operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats, treated with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

First experiment (dose-finding study, all strains): 4.88 - 5000 µg/plate with and without metabolic activation (tested up to the limit concentration)
Second experiment (main assay, all strains): 156 - 5000 µg/plate with and without metabolic activation (tested up to the limit concentration)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solvent was chosen based on its solubiulity properties and its nontoxicity for the bacteria.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

sterilized purified water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: The dose-finding assay and main assay were carried out in duplicate plating for each dose level.

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial plate

Evaluation criteria:

Acceptance criteria
When at least one plate count in the solvent and positive control group is within the acceptance control range (mean ± 3SD, calculated from the historical control data, see table 3), the assay is judged to be acceptable.

Evaluation criteria
When the test substance shows a dose-dependent increase in the number of revertant colonies to at least twice as many as that of the solvent control, the response is judged to be positive 4 5). The study director judges the result with scientific consideration of biological relevance. When the positive response is reproducible, the test substance is judged to have mutagenic potential.

Statistics:

No statistics were performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

decrease in number of revertants in exp. 2, starting at 1250 µg/mL, with S9 mix (around 32-45%)

Vehicle controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

number of revertants is close to or within historical control date range

Any other information on results incl. tables

Table 1: Summary of test results (experiment 1; dose-finding assay)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Frameshift type		Base-pair substitution type
		TA98	TA1537	TA100	TA1535	WP2 uvrA
–	Solvent control (water)	20	7	104	9	22
–	4.88	21	7	110	8	19
–	19.5	23	4	105	7	23
–	78.1	20	9	106	8	19
–	313	15	6	102	5	14
–	1250	18	9	108	11	21
–	5000	15	5	111	13	19
–	Positive controls (concentrations (µg/plate))	AF-2 (0.1)	9AA (80)	AF-2 (0.01)	SA (0.5)	AF-2 (0.01)
	Mean No. of colonies/plate (average of 2 plates)	432	341	646	306	107
+	Solvent control (water)	29	15	104	6	24
+	4.88	34	14	110	10	21
+	19.5	34	14	126	8	18
+	78.1	32	13	115	7	26
+	313	43	13	111	9	21
+	1250	36	13	112	7	24
+	5000	46	10	126	7	18
+	Positive controls (concentrations (µg/plate))	2AA (0.5)	2AA (2)	2AA (1)	2AA (2)	2AA (10)
	Mean No. of colonies/plate (average of 2 plates)	259	147	708	206	264

SA = sodium azide

AF-2=2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

9AA= 9-Aminoacridine

2AA = 2-Aminoanthracene

SD = standard deviation

Table 2: Summary of test results (experiment 2; main assay)

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 2 plates)
		Frameshift type		Base-pair substitution type
		TA98	TA1537	TA100	TA1535	WP2 uvrA
–	Solvent control (water)	20	11	106	10	21
–	156	29	14	114	11	27
–	313	27	11	106	12	16
–	625	27	10	112	13	22
–	1250	23	6	113	12	17
–	2500	30	11	110	7	23
–	5000	27	15	105	9	22
–	Positive controls (concentrations (µg/plate))	AF-2 (0.1)	9AA (80)	AF-2 (0.01)	SA (0.5)	AF-2 (0.01)
	Mean No. of colonies/plate (average of 2 plates)	370	388	633	347	84
+	Solvent control (water)	43	22	115	12	24
+	156	43	19	109	9	23
+	313	42	19	119	9	18
+	625	43	22	119	14	23
+	1250	41	15	115	9	24
+	2500	43	16	119	8	23
+	5000	44	12	126	14	31
+	Positive controls (concentrations (µg/plate))	2AA (0.5)	2AA (2)	2AA (1)	2AA (2)	2AA (10)
	Mean No. of colonies/plate (average of 2 plates)	249	137	727	206	297

SA = sodium azide

AF-2 =2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide

9AA= 9-Aminoacridine

2AA = 2-Aminoanthracene

SD = standard deviation

Table 3: Historical control data (Jan - Dec 2013)

		Revertant colonies
Test strain	S9 Mix	Solvent control		Positive control^(1)
		Mean ± SD	Number of data	Mean ± SD	Number of data
TA100	-	90.7 ± 8.93	203	574.5 ± 68.48	199
TA1535	-	10.5 ± 2.56	162	287.4 ± 40.55	162
WP2 uvrA	-	18.1 ± 3.91	159	96.4 ± 13.59	159
TA98	-	20.7 ± 3.95	158	353.0 ± 34.85	158
TA1537	-	9.0 ± 2.69	162	445.2 ± 75.08	162
TA100	+	92.2 ± 10.32	193	566.1 ± 55.17	193
TA1535	+	10.3 ± 2.54	157	165.5 ± 16.44	157
WP2 uvrA	+	21.6 ± 4.69	161	270.3 ± 49.98	161
TA98	+	30.7 ± 5.29	196	192.6 ± 23.40	192
TA1537	+	15.7 + 3.93	157	99.9 ± 18.23	157

Mean ± 3SD were set as the acceptable control range.

 

(1) Positive control substances

Test strain	S9 Mix	Chemical	Dose [µg/plate]
TA100	-	2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide	0.01
TA1535	-	sodium azide	0.5
WP2 uvrA	-	2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide	0.01
TA98	-	2-(2-fury 1)-3-(5-nitro-2-fury 1) acrylamide	0.1
TA1537	-	9-aminoacridine	80
TA100	+	2-aminoanthracene	1
TA1535	+		2
WP2 uvrA	+		10
TA98	+		0.5
TA1537	+		2