Tetraol

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:

2014-11-26 to 2014-12-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 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 (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 003
- Expiration date of the lot/batch: 01 Jan 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, exclusion of direct sunlight, test substance is hygroscopic
- Stability under test conditions: stable

Method

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:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (phenobarbital and β-naphthoflavone induced rat liver)

Test concentrations with justification for top dose:

0; 33; 100; 333; 1 000; 2 700 and 5400 μg/plate

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2-AA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix (all tester strains tested)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

MNNG

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

without S9 mix; 5 µg/plate (TA1535, TA100)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NOPD

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

without S9 mix; 10 μg/plate (TA 98)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

AAC

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix; 100 µg/plate (TA1537)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

4-NQO

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix; 5 µg/plate (E. coli WP2 uvrA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (SPT); preincubation (PIT)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 hours in the dark at 37 °C

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of his+ or trp+ revertants

Evaluation criteria:

Acceptance criteria
Generally, the experiment was considered valid if the following criteria were met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used.

Assessment criteria
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA98, TA100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA1535 and TA1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

Results and discussion

Test results

Key result

Species / strain:

bacteria, other: TA98, TA100, TA1535, TA1537, E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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: No
- Effects of osmolality: No
- Evaporation from medium: No
- Water solubility: No
- Precipitation:

RANGE-FINDING/SCREENING STUDIES:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]

Any other information on results incl. tables

Table 1 Raw data

Experiment 1 (SPT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results with S9
DMSO	12	9.7	31	37	66.7
33	11.7	9.3	27.7	32.3	66.7
100	9.3	8.7	33.7	38	63
333	12.7	8.7	30.3	37.7	64.7
1000	11.3	8	26.7	29	71.3
2700	11.7	3	30.7	43.7	60.3
5400	11	4	22	34.3	52.7
positive control	202	140.3	1672.7	746.3	206
	Results without S9
DMSO	11.7	7.3	25	38.7	65
33	10.3	7.3	25	35.7	60.3
100	12.3	10	23	40.3	61.3
333	12	7.3	22.7	31.3	59.7
1000	12	11	27.3	34	47
2700	15	6.3	20.7	42.3	48
5400	10.7	1.7	20.3	24.3	38.7
positive control	4552	697.7	444	4474	900.3
Experiment 2 (PIT)
Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates with different strains of Salmonella typhimurium and Escherichia coli
	TA1535	TA1537	TA98	TA100	WP2 uvrA
	Results with S9
DMSO	9	10.3	28.3	28.3	86.7
33	9.3	7.3	32.3	29.7	88
100	9.3	7.3	30	27.3	86.3
333	9.7	6.7	25.7	29	94.3
1000	7.7	6.3	35	30.7	88.3
2700	9	5.7	19	27.3	97.7
5400	7.3	6	21	29.7	79
positive control	179.3	116.7	1276.3	663	208.7
	Results without S9
DMSO	11.3	7.3	21	31.7	99.7
33	9.7	7.7	20.3	27	90.3
100	12	5.3	19.7	28.7	97
333	8	4.3	22.3	25.3	79.7
1000	9.3	8.3	16	23.3	92
2700	8.7	4.3	21	24.3	75.7
5400	7	4.7	19.3	29	89.7
positive control	1568.3	591.3	354.3	2092.3	253