Butanal, reaction products with N2,N2'-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-1,3,5-triazine-2,4,6-triamine and hydrogen peroxide

Genetic toxicity: in vitro

Administrative data

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:

16 Feb 2012 - 02 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP (2-AA was the only positive control used with metabolic activation).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

2-AA was the only positive control used for metabolic activation

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

GSID 3056-1

IUPAC Name:

GSID 3056-1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): GSID 3056-1
- Physical state: solid, brownish
- Analytical purity: 98% (Analytical Report 11L00523)
- Lot/batch No.: 1470 VB04
- Storage condition of test material: room temperature
- Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 06 Oct 2013 (as indicated by the sponsor, and the sponsor holds this responsibility).

Method

Target gene:

his operon for S. typhimurium strains
trp operon for the E. coli strain

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from Wistar rats treated with 80 mg/kg bw phenobarbital i.p. and β-naphthoflavone orally each on three consecutive days.

Test concentrations with justification for top dose:

First experiment (standard plate test, with and without metabolic activation, triplicate): 0, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation, triplicate): 0, 10, 33, 100, 333, 1000 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Due to the limited solubility of the test substance in ultrapure water, acetone was used as vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2.5 µg/plate, in DMSO, for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate, in DMSO, for E. coli WP2 uvrA

Controls 2

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

5 µg/plate, in DMSO, for TA 1535 and TA 100

Controls 3

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate, in DMSO, for TA 1537

Migrated to IUCLID6: (AAC)

Controls 4

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

5 µg/plate, in DMSO, for E. coli WP2 uvrA

Migrated to IUCLID6: (4-NQO)

Controls 5

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

10 µg/plate, in DMSO, for TA 98

Details on test system and experimental conditions:

In the standard plate test, tubes were filled with 2mL portions of soft agar and kept in a water bath at 42-45°C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples with S. typhimurium strains were were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37°C.
After mixing samples with the E.coli train were poured onto SA1 selective agar plates ( minimal agar prepared according to Green and Muriel, Mut Res 38: 3-32, 1976) and incubated for 48 - 72 hrs in the dark at 37°C.

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of the S9 mix were incubated at 37°C for 20 minutes (Yahagi et al., Mut Res 48: 121-129,1977; Matsushima et al. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York,1980). After addition of 2 mL soft agar samples were poured onto agar plates and incubated again at 37°C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.
Triplicate testing was done.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+9/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in number of revertants was observed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Decrease in the number of his+ or trp+ revertants, slight reduction in the titer observed depending on the strain and test conditions from about 2 500 μg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in number of revertants was observed.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, slight reduction in the titer, observed depending on the strain and test conditions from about 1 000 μg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation:
Precipitation of the test substance was observed in the standard plate test from 333 μg/plate onward (without S9 mix) and from 1000 μg/plate onward (with S9 mix).
Precipitation of the test substance was observed in the preincubation test from 1000 μg/plate onward with and without S9 mix.

Remarks on result:

other: other: Standard Plate Test

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1: Results of Experiment I, Standard Plate Test

With or	Test substance	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
without	concentration	Base-pair substitution type			Frameshift type
S9 mix	(µg/plate)	TA100	TA1535	E.coli WP2	TA98	TA1537
-	acetone	68 ± 7	12 ± 2	44 ± 3	19 ± 3	6 ± 1
-	33	78 ± 8	11 ± 2	43 ± 3	20 ± 3	6 ± 1
-	100	79 ± 6	12 ± 1	42 ± 11	17 ± 2	7 ± 2
-	333	75 ± 10	12 ± 2	41 ± 4	19 ± 2	6 ± 1
-	1000	69 ± 6	11 ± 2	40 ± 7	17 ± 2	5 ± 2
-	2500	40 ± 12	7 ± 1	28 ± 2	12 ± 4	3 ± 2
-	5000	6 ± 1	4 ± 2	21 ± 6	3 ± 1	1 ± 0
positive controls, -S9	Name	MNNG	MNNG	4 -NQO	NOPD	AAC
	Concentration [µg/plate]	5.0	5.0	5.0	10	2.5
	Mean No. of colonies/plate (average of 3 ± SD)	672 ± 93	637 ± 23	650 ± 44	846 ± 79	410 ± 20
		TA100	TA1535	E.coli WP2	TA98	TA1537
+	acetone	79 ± 7	13 ± 1	48 ± 3	22 ± 3	7 ± 1
+	33	81 ± 7	13 ± 3	44 ± 10	28 ± 4	7 ± 1
+	100	77 ± 2	12 ± 1	48 ± 2	22 ± 2	9 ± 0
+	333	78 ± 10	13 ± 2	47 ± 7	25 ± 4	6 ± 1
+	1000	81 ± 4	12 ± 2	45 ± 6	21 ± 5	7 ± 1
+	2500	58 ± 21	5 ± 2	35 ± 7	16 ± 2	3 ± 1
+	5000	15 ± 7	2 ± 2	29 ± 3	7 ± 2	1 ± 1
positive controls, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Concentration [µg/plate]	2.5	2.5	60	2.5	100
	Mean No. of colonies/plate (average ± SD)	757 ± 43	126 ± 18	261 ± 28	651 ± 63	105 ± 5

Table2: Results of Experiment II, Preincubation Test

With or	Test substance	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
without	concentration	Base-pair substitution type			Frameshift type
S9 mix	(µg/plate)	TA100	TA1535	E.coli WP2	TA98	TA1537
-	acetone	74 ± 6	12 ± 2	44 ± 4	18 ± 3	7 ± 1
-	10	79 ± 8	11 ± 2	42 ± 8	18 ± 4	7 ± 2
-	33	72 ± 1	12 ± 4	38 ± 5	18 ± 7	8 ± 2
-	100	73 ± 3	12 ± 1	42 ± 6	17 ± 3	6 ± 2
-	333	72 ± 10	13 ± 2	43 ± 6	16 ± 2	7 ± 1
-	1000	61 ± 11	9 ± 2	34 ± 6	11 ± 3	5 ± 3
-	2500	B/P	B/P	13 ± 3	B/P	B/P
positive controls, -S9	Name	MNNG	MNNG	4 -NQO	NOPD	AAC
	Concentration [µg/plate]	5.0	5.0	5.0	10	100
	Mean No. of colonies/plate (average of 3 ± SD)	672 ± 93	721 ± 48	591 ± 39	566 ± 33	359 ± 34
		TA100	TA1535	E.coli WP2	TA98	TA1537
+	acetone	82 ± 10	13 ± 3	44 ± 9	27 ± 6	9 ± 1
+	10	88 ± 14	11 ± 1	44 ± 12	28 ± 8	9 ± 1
+	33	88 ± 7	13 ± 4	47 ± 8	28 ± 12	8 ± 3
+	100	81 ± 8	14 ± 2	44 ± 7	29 ± 7	9 ± 3
+	333	82 ± 11	12 ± 3	50 ± 4	29 ± 6	8 ± 1
+	1000	70 ± 6	13 ± 2	31 ± 9	25 ± 4	8 ± 4
+	2500	52 ± 6	8 ± 2	18 ± 4	19 ± 3	6 ± 1
positive controls, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Concentration [µg/plate]	2.5	2.5	60	2.5	2.5
	Mean No. of colonies/plate (average ± SD)	860 ± 67	136 ± 19	258 ± 17	599 ± 47	120 ± 13

P = Precipitation; B = Reduced Background growth

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

CLP: not classified
DSD: not classified