AERO 5100 PROMOTER

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:

1 - 8 June 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US EPA 40 CFR 799 (1997) Toxic Substances Control Act Test Guidelines - Sub-section 799.9510, TSCA bacterial reverse mutation test. Federal Register, Vol. 62, No. 158

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

434-440-3

EC Name:

-

Cas Number:

86329-09-1

Molecular formula:

C8H15NSO

IUPAC Name:

O-(2-methylpropyl) prop-2-en-1-ylcarbamothioate

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (single i.p. injection of 500 mg/kg bw)

Test concentrations with justification for top dose:

Experiment 1: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: ethanol
- Justification for choice of solvent/vehicle: the test substance was soluble in the vehicle at 50 mg/mL

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG), 9-aminoacridine (9-AA), 2-nitrofluorene (2-NF), 2-aminoanthracene (2-AA), benzo[a]pyrene (BaP)

Remarks:

+S9: 2-AA (2 µg/plate, TA1535; 10 µg/plate, WP2uvrA/pKM101); BaP (5 µg/plate, TA1537, TA98, TA100) -S9: ENNG (5 µg/plate, TA1535; 3 µg/plate, TA100; 2 µg/plate, WP2uvrA/pKM101); 9-AA (80 µg/plate, TA1537); 2-NF (1 µg/plate, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: ca. 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in revertant colony numbers or thinning of the bacterial background lawn

Evaluation criteria:

The mutagenic activity of a test substance was assessed by applying the following criteria:
1) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
2) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls in either mutation test it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
3) If the results obtained fail to satisfy the criteria for a clear positive or negative response given paragraphs 1) and 2), additional testing may be performed in order to resolve the issue of the test substance’s mutagenic activity in this test system. Should an increase in revertant colony numbers then be observed which satisfies paragraph 1) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA 1537, TA98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in TA1535 and TA100 with S9 mix

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in all strains at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Table 1. Test results of Experiment 1.

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA/ pKM101	TA1537	TA98
-	0 (EtOH)	19 ± 4	98 ± 15	101 ± 7	13 ± 3	40 ± 1
-	5	15 ± 2	89 ± 5	88 ± 15	10 ± 4	36 ± 7
-	15	15 ± 2	97 ± 9	102 ± 5	12 ± 2	44 ± 9
-	50	14 ± 2	96 ± 7	121 ± 3	12 ± 2	32 ± 5
-	150	14 ± 1	89 ± 7	106 ± 11	11 ± 1	41 ± 9
-	500	16 ± 3	89 ± 6	111 ± 9	12 ± 2	39 ± 6
-	1500	13 ± 1	94 ± 15	100 ± 6	11 ± 2	32 ± 5
-	5000	8 ± 4^T	62 ± 6^T	61 ± 15^T	3 ± 0^T	20 ± 2^T
Positive controls, –S9	Name	ENNG	ENNG	ENNG	9-AA	2-NF
	Concentration [μg/plate]	5	3	2	80	1
	Mean No. of colonies/plate (average of 3 ± SD)	148± 15	437± 20	2312± 48	925± 149	273± 48
+	0 (EtOH)	16± 3	97± 4	104± 10	14± 1	38±1
+	5	16± 3	100± 10	100± 9	14± 2	43± 7
+	15	25± 2	97± 3	96± 16	11± 1	40± 1
+	50	35± 4	116 ± 2	89± 3	9± 1	37± 1
+	150	76± 5	141± 14	90± 10	10± 2	38± 6
+	500	125± 6	165± 1	95± 2	9± 1	37± 3
+	1500	211± 26	171± 3	105± 9	10± 3	34 ± 5
+	5000	25± 1^T	56± 11^T	53± 11^T	6± 1^T	20± 2^T
Positive controls, +S9	Name	2-AA	BaP	2-AA	BaP	BaP
	Concentration [μg/plate]	2	5	10	5	5
	Mean No. of colonies/plate (average of 3 ± SD)	381± 45	492± 28	613± 13	215± 30	685± 33

EtOH: ethanol

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

BaP: benzo[a]pyrene

T: thinning of bacterial background lawn

 

Table 2. Test results of Experiment 2.

With or without S9-Mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA1535	TA100	WP2 uvrA/ pKM101	TA1537	TA98
-	0 (EtOH)	17 ± 2	107 ± 10	110 ± 4	14 ± 1	38 ± 5
-	50	17 ± 3	85 ± 19	95 ± 11	14 ± 1	40 ± 5
-	150	13 ± 4	90 ± 5	100 ± 14	11 ± 2	41 ± 4
-	500	13 ± 2	83 ± 8	96 ± 8	12 ± 2	36 ± 3
-	1500	12 ± 4	66 ± 7	90 ± 10	11 ± 2	34 ± 5
-	5000	5 ± 1^T	49 ± 7^T	37 ± 7^T	6 ± 1^T	9 ± 3^T
Positive controls, –S9	Name	ENNG	ENNG	ENNG	9-AA	2-NF
	Concentration [μg/plate]	5	3	2	80	1
	Mean No. of colonies/plate (average of 3 ± SD)	219 ± 18	437 ± 37	1787 ± 63	1324 ± 202	228 ± 5
+	0 (EtOH)	18± 4	101± 8	107± 12	13± 1	38± 1
+	50	26± 3	90± 11	103± 15	7± 2	35± 7
+	150	58± 5	138± 2	110± 12	7± 1	39± 6
+	500	93± 5	151± 4	113± 15	10± 2	36± 5
+	1500	143± 10	211± 21	107± 11	10± 2	33± 4
+	5000	14± 2^T	46± 5^T	72± 10^T	6± 0^T	11± 4^T
Positive controls, +S9	Name	2-AA	BaP	2-AA	BaP	BaP
	Concentration [μg/plate]	2	5	10	5	5
	Mean No. of colonies/plate (average of 3 ± SD)	495 ± 16	650 ± 30	813 ± 90	185 ± 22	497 ± 28

EtOH: ethanol

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

9-AA: 9-aminoacridine

2-NF: 2-nitrofluorene

BaP: benzo[a]pyrene

T: thinning of bacterial background lawn

In Experiment 1 and 2, substantial increases in revertant colony numbers over control counts were observed in TA1535 following exposure to the test substance with metabolic activation. Small increases (< 2-fold) and substantial increased in revertant colony numbers over control counts were obtained in strain TA100 with metabolic activation in Experiment 1 and 2, respectively. In both strains increases were maximal at 1500 μg/plate and a clear dose-response relationship was observed.

Appropriate positive control substances induced substantial increases in revertant colony numbers with all strains, confirming sensitivity of the cultures and activity of the S9 mix.

Applicant's summary and conclusion

Conclusions:

Under the conditions of the Ames test the substance was mutagenic in strains TA 1535 and TA 100 tested with metabolic activation and non mutagenic without meatbolic activation, while in strains TA 1537, TA 98 and WP2 uvr A pKM 101 it was non mutagenic with and without metabolic activation.