4,4'-dioxo-4,4'-dioxydibutyric acid

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:

05 February 2018 - 23 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-dioxo-4,4'-dioxydibutyric acid

EC Number:

204-611-5

EC Name:

4,4'-dioxo-4,4'-dioxydibutyric acid

Cas Number:

123-23-9

Molecular formula:

C8H10O8

IUPAC Name:

4-[(3-carboxypropanoyl)peroxy]-4-oxobutanoic acid

Test material form:

liquid

Specific details on test material used for the study:

Batch No.: 000071307
Purity: 69.9%

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 97a, TA 98, TA 100, TA102 and TA 1535,

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9 mix

Test concentrations with justification for top dose:

Concentrations:
- Experiment 1a: 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 1b: TA97a and TA102: 15, 50, 150, 500, 1500 and 5000 µg/plate; TA98 and TA100: 0.5, 1.5, 5, 15, 50, and 150 µg/plate; TA1535: 5, 15, 50, 150, 500 and 1500 µg/plate
- Experiment 2: TA97a and TA102: 39, 78, 156, 313, 625, 1250, 2500 and 5000 µg/plate; TA98 and TA100: 1.2, 2.3, 4.7, 9.4, 18.8, 37.5, 75 and 150 µg/plate; TA1535: 12, 23, 47, 94, 188, 375, 750 and 1500 µg/plate;
Justification for top dose: tested up to recommended maximum test concentration according to OECD 471 in experiment 1a; due to observed toxicity lower concentrations were chosen for the experiment 1b and 2

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined. Based on these results, DMSO was chosen as solvent.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and demineralised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: Nitrophenylendiamine and 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1a and 1b: in agar (plate incorporation); experiment 2: preincubation

DURATION
- Preincubation period: 20-30 minutes, 37 °C
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: determination revertant colony number and

Evaluation criteria:

The mean values and standard deviations of each threefold determination are calculated as well as the increase factor of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (mean revertants minus mean spontaneous revertants) is given. A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed . A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Statistics:

Not performed as not mandatory for this test system.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 97

Remarks:

TA97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed

Any other information on results incl. tables

Table 1: Mean Revertants Experiment 1a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	69	66	32	27	78	72	225	263	21	19
	sd	16.2	6.0	2.6	3.5	15.4	10.0	19.6	56.0	1.5	3.2
DMSO	Mean	69	95	27	28	73	78	225	287	19	21
	sd	10.4	14.0	6.0	1.2	12.0	9.5	16.7	18.9	3.8	3.6
Positive Controls*	Mean	329	657	s.g.	101	323	s.g.	692	727	257	143
	sd	99.6	79.4	0.0	24.6	54.5	0.0	36.0	47.4	14.0	23.0
	f(I)	4.77	6.92	37.07	3.61	4.14	12.83	3.08	2.53	12.24	6.81
5000 µg/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
	Background lawn	-	-	-	-	-	-	-	-	-	-
1500 µg/plate	Mean	73	79	0	0	27	46	201	256	12	15
	sd	10.6	12.6	0.0	0.0	5.9	4.4	32.3	4.0	1.0	4.4
	f(I)	1.06	0.83	0.00	0.00	0.37	0.59	0.89	0.89	0.63	0.71
	Background lawn	++	++	++	++	++	++	++	++	++	++
500 µg/plate	Mean	89	73	1	2	25	35	256	228	19	19
	sd	20.4	6.7	0.0	1.2	3.0	4.4	13.9	44.0	3.6	4.2
	f(I)	1.29	0.77	0.04	0.07	0.34	0.45	1.14	0.79	1.00	0.90
	Background lawn	++	++	++	++	++	++	++	++	++	++
150 µg/plate	Mean	74	82	3	4	33	44	247	252	22	16
	sd	10.6	19.2	3.5	1.7	7.5	1.5	38.9	32.7	1.2	1.2
	f(I)	1.07	0.86	0.11	0.14	0.45	0.56	1.10	0.88	1.16	0.76
	Background lawn	++	++	++	++	++	++	++	++	++	++
50 µg/plate	Mean	78	110	28	31	82	78	213	243	21	18
	sd	10.4	21.4	3.8	2.9	5.9	10.4	36.3	28.1	5.5	2.5
	f(I)	1.13	1.16	1.04	1.11	1.12	1.00	0.95	0.85	1.11	0.86
	Background lawn	++	++	++	++	++	++	++	++	++	++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,s.g. = strong growth, too strong for counting of revertants

 

Table 2: Mean Revertants Experiment 1b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	73	84	36	33	92	108	249	259	21	22
	sd	7.6	5.1	6.2	5.5	8.0	15.9	26.6	53.3	1.2	1.5
DMSO	Mean	74	110	38	36	89	91	239	249	26	18
	sd	5.2	21.1	4.4	11.9	9.2	1.2	28.9	46.0	1.7	0.6
Positive Controls*	Mean	479	463	268	176	379	s.g.	657	1323	220	175
	sd	67.2	132.3	28.0	24.3	62.3	0.0	9.2	60.0	10.6	31.1
	f(I)	6.47	4.21	7.05	4.89	4.12	11.00	2.75	5.31	10.48	9.72
5000 µg/plate	Mean	6	55	n.d.	n.d.	n.d.	n.d.	0	0	n.d.	n.d.
	sd	5.8	11.9	n.d.	n.d.	n.d.	n.d.	0.0	0.0	n.d.	n.d.
	f(I)	0.08	0.50	n.d.	n.d.	n.d.	n.d.	0.00	0.00	n.d.	n.d.
	Background lawn	++	++					-	-	n.d.	n.d.
1500 µg/plate	Mean	76	91	n.d.	n.d.	n.d.	n.d.	221	213	3	3
	sd	5.5	22.0	n.d.	n.d.	n.d.	n.d.	4.6	24.1	1.7	1.2
	f(I)	1.03	0.83	n.d.	n.d.	n.d.	n.d.	0.92	0.86	0.12	0.17
	Background lawn	++	++					++	++	++	++
500 µg/plate	Mean	74	87	n.d.	n.d.	n.d.	n.d.	227	251	20	17
	sd	11.1	17.1	n.d.	n.d.	n.d.	n.d.	12.9	48.1	2.1	0.6
	f(I)	1.00	0.79	n.d.	n.d.	n.d.	n.d.	0.95	1.01	0.77	0.94
	Background lawn	++	++					++	++	++	++
150 µg/plate	Mean	77	93	6	7	0	0	223	199	19	17
	sd	11.5	9.5	2.9	1.7	0.0	0.0	14.0	9.2	3.5	0.0
	f(I)	1.04	0.85	0.16	0.19	0.00	0.00	0.93	0.80	0.73	0.94
	Background lawn	++	++	++	++	-	-	++	++	++	++
50 µg/plate	Mean	96	116	32	35	102	102	220	196	20	16
	sd	13.4	7.2	4.4	5.8	18.5	12.2	20.8	21.2	3.0	4.0
	f(I)	1.30	1.05	0.84	0.97	1.15	1.12	0.92	0.79	0.77	0.89
	Background lawn	++	++	++	++	++	++	++	++	++	++
15 µg/plate	Mean	77	101	37	37	75	92	203	229	17	18
	sd	14.0	10.3	2.5	5.2	9.2	8.7	20.5	30.0	1.0	2.0
	f(I)	1.04	0.92	0.97	1.03	0.84	1.01	0.85	0.92	0.65	1.00
	Background lawn	++	++	++	++	++	++	++	++	++	++
5 µg/plate	Mean	n.d.	n.d.	30	33	95	93	n.d.	n.d.	19	17
	sd	n.d.	n.d.	3.5	1.7	18.9	9.5	n.d.	n.d.	3.6	2.1
	f(I)	n.d.	n.d.	0.79	0.92	1.07	1.02	n.d.	n.d.	0.73	0.94
	Background lawn			++	++	++	++			++	++
1.5 µg/plate	Mean	n.d.	n.d.	32	34	101	106	n.d.	n.d.	n.d.	n.d.
	sd	n.d.	n.d.	8.1	3.5	5.0	11.9	n.d.	n.d.	n.d.	n.d.
	f(I)	n.d.	n.d.	0.84	0.94	1.13	1.16	n.d.	n.d.	n.d.	n.d.
	Background lawn			++	++	++	++
0.5 µg/plate	Mean	n.d.	n.d.	34	36	99	101	n.d.	n.d.	n.d.	n.d.
	sd	n.d.	n.d.	2.5	2.9	9.9	4.2	n.d.	n.d.	n.d.	n.d.
	f(I)	n.d.	n.d.	0.89	1.00	1.11	1.11	n.d.	n.d.	n.d.	n.d.
	Background lawn			++	++	++	++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, n.d. = not determined, due to the toxicity results of the experiment 1a, f(I) = increase factor,s.g. = strong growth, too strong for counting of revertants. * Different positive controls were used

 

Table 3: Mean Revertants Experiment 2 (TA97a, TA102)

Strain		TA97a		TA102
Induction		-S9	+S9	-S9	+S9
Demin. water	Mean	79	76	308	284
	sd	3.2	11.3	35.6	30.2
DMSO	Mean	79	95	308	261
	sd	15.9	5.8	55.6	64.0
Positive Controls*	Mean	577	433	1339	1181
	sd	182.5	32.3	72.6	257.2
	f(I)	7.30	4.56	4.35	4.52
5000 µg/plate	Mean	4	14	33	29
	sd	1.0	2.1	12.4	21.4
	f(I)	0.05	0.15	0.11	0.11
	Background lawn	++	++	++	++
2500 µg/plate	Mean	18	27	229	295
	sd	7.4	7.6	47.7	26.6
	f(I)	0.23	0.28	0.74	1.13
	Background lawn	++	++	++	++
1250 µg/plate	Mean	82	80	233	331
	sd	0.6	7.5	32.3	61.2
	f(I)	1.04	0.84	0.76	1.27
	Background lawn	++	++	++	++
625 µg/plate	Mean	80	84	259	347
	sd	13.9	7.1	18.9	57.7
	f(I)	1.01	0.88	0.84	1.33
	Background lawn	++	++	++	++
313 µg/plate	Mean	79	85	321	273
	sd	3.5	18.6	2.3	45.5
	f(I)	1.00	0.89	1.04	1.05
	Background lawn	++	++	++	++
156 µg/plate	Mean	77	76	301	240
	sd	6.7	9.6	35.9	4.0
	f(I)	0.97	0.80	0.98	0.92
	Background lawn	++	++	++	++
78 µg/plate	Mean	71	67	264	263
	sd	11.3	2.5	0.0	39.5
	f(I)	0.90	0.71	0.86	1.01
	Background lawn	++	++	++	++
39 µg/plate	Mean	71	84	261	241
	sd	10.6	25.2	24.1	24.1
	f(I)	0.90	0.88	0.85	0.92
	Background lawn	++	++	++	++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

Table 4: Mean Revertants Experiment 2 (TA98, TA100)

Strain		TA98		TA100
Induction		-S9	+S9	-S9	+S9
Demin. water	Mean	37	37	85	93
	sd	5.9	8.7	9.5	12.5
DMSO	Mean	37	42	75	84
	sd	3.6	3.1	18.9	7.6
Positive Controls*	Mean	621	264	699	717
	sd	56.8	18.3	25.7	37.0
	f(I)	16.78	6.29	8.22	8.54
150 µg/plate	Mean	3	5	15	16
	sd	0.6	1.7	3.1	2.6
	f(I)	0.08	0.12	0.20	0.19
	Background lawn	++	++	++	++
75 µg/plate	Mean	34	37	88	78
	sd	3.5	9.3	6.4	3.1
	f(I)	0.92	0.88	1.17	0.93
	Background lawn	++	++	++	++
37.5 µg/plate	Mean	44	44	76	86
	sd	4.7	4.5	7.6	5.8
	f(I)	1.19	1.05	1.01	1.02
	Background lawn	++	++	++	++
18.8 µg/plate	Mean	40	30	84	72
	sd	5.6	7.8	4.0	1.7
	f(I)	1.08	0.71	1.12	0.86
	Background lawn	++	++	++	++
9.4 µg/plate	Mean	42	46	82	77
	sd	1.0	12.9	1.7	4.6
	f(I)	1.14	1.10	1.09	0.92
	Background lawn	++	++	++	++
4.7 µg/plate	Mean	38	37	76	77
	sd	4.6	9.3	7.2	3.8
	f(I)	1.03	0.88	1.01	0.92
	Background lawn	++	++	++	++
2.3 µg/plate	Mean	36	35	82	87
	sd	5.0	8.1	6.9	8.7
	f(I)	0.97	0.83	1.09	1.04
	Background lawn	++	++	++	++
1.2 µg/plate	Mean	38	33	92	87
	sd	10.3	3.8	13.7	3.1
	f(I)	1.03	0.79	1.23	1.04
	Background lawn	++	++	++	++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

 

Table 5: Mean Revertants Experiment 2 (TA1535)

Strain		TA1535
Induction		-S9	+S9
Demin. water	Mean	10	12
	sd	2.1	3.5
DMSO	Mean	11	12
	sd	2.5	4.0
Positive Controls*	Mean	249	113
	sd	72.0	29.5
	f(I)	24.90	9.42
1500 µg/plate	Mean	2	3
	sd	0.6	1.2
	f(I)	0.18	0.25
	Background lawn	++	++
750 µg/plate	Mean	12	13
	sd	0.6	1.2
	f(I)	1.09	1.08
	Background lawn	++	++
375 µg/plate	Mean	12	12
	sd	1.7	1.2
	f(I)	1.09	1.00
	Background lawn	++	++
188 µg/plate	Mean	12	15
	sd	2.1	2.1
	f(I)	1.09	1.25
	Background lawn	++	++
94 µg/plate	Mean	12	13
	sd	1.2	3.2
	f(I)	1.09	1.08
	Background lawn	++	++
47 µg/plate	Mean	9	10
	sd	1.5	1.5
	f(I)	0.82	0.83
	Background lawn	++	++
23 µg/plate	Mean	11	9
	sd	3.5	1.5
	f(I)	1.00	0.75
	Background lawn	++	++
12 µg/plate	Mean	11	13
	sd	1.5	1.2
	f(I)	1.00	1.08
	Background lawn	++	++

++= background lawn, + = background lawn reduced, - = no background lawn, sd = standard deviation, f(I) = increase factor,* Different positive controls were used

 

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

A study according to OECD guideline 471 was performed to assess the genotoxic potential of the test item. The test item was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

In the experiment 1a, the test item (dissolved in DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. Signs of toxicity towards the bacteria strains (no bacterial background lawn and/or no revertant growth) were observed in both the absence and presence of metabolic activation from 150 µg/plate upwards depending on the strain used. Due to the toxicity results in the experiment 1a, in the experiemnt 1b and 2the test item (dissolved in DMSO) was tested up to the following concentrations in the absence and presence of S9-mix: 150 µg/plate (TA98, TA100), 1500 µg/plate (TA1535) and 5000 µg/plate (TA97a, TA102). The test item showed no precipitates on the plates at any of the concentrations. At the concentration 150 µg/plate and upwards (depending on the strain used), signs of toxicity were observed towards the respective bacteria strain with and without metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.