Orthoperiodic 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:

01 January 2018 to 22 January 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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Orthoperiodic acid

EC Number:

233-937-0

EC Name:

Orthoperiodic acid

Cas Number:

10450-60-9

Molecular formula:

H5IO6

IUPAC Name:

tetrahydroxyiodous acid

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: White crystalline powder
- Storage: At room temperature

Method

Target gene:

- Histidine requirement in the Salmonella typhimurium strains (Histidine operon).
- Tryptophan requirement in the Escherichia coli strain (Tryptophan operon).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The Salmonella typhimurium strains are regularly checked to confirm their histidine requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV sensitivity and the number of spontaneous revertants. Stock cultures of the strains were stored in liquid nitrogen (-196 °C).

MEDIA USED
- Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per litre: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine

- Top agar: Milli-Q water containing 0.6 % (w/v) bacteriological agar and 0.5 % (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1 °C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (10^9 cells/mL). Freshly grown cultures of each strain were used for testing.
- Properly maintained: Yes. The strain is regularly checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants. Stock cultures were stored in liquid nitrogen (-196 °C).

MEDIA USED
Agar plates
Agar plates (ø 9 cm) containing 25 mL glucose agar medium. Glucose agar medium contained per litre: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan.

Top agar
Milli-Q water containing 0.6 % (w/v) bacteriological agar and 0.5 % (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3 °C.

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by Aroclor 1254)

Test concentrations with justification for top dose:

- Dose range finding study (TA100 and WP2uvrA only): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (with and without S9-mix)
- Experiment 1 (TA1535, TA1537 and TA98): 52, 164, 512, 1600 and 5000 μg/plate (with and without S9-mix)
- Experiment 2 (TA100, TA1535 and WP2uvrA): 52, 164, 512, 1600 and 5000 μg/plate (TA100: with and without S9-mix, TA1535 and WP2uvrA: with S9-mix only)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Milli-Q water
- Justification for choice of solvent/vehicle: A solubility test was performed based on visual assessment. The test material was dissolved in Milli-Q water.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DOSE RANGE FINDING TEST/ MUTATION ASSAY
- Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate were tested in triplicate.
The highest concentration of the test material used in the subsequent mutation assays was 5000 µg/plate. At least five different doses (increasing with approximately half-log steps) of the test material were tested in triplicate in each strain in the absence and presence of S9-mix.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

MUTATION ASSAY
- The above mentioned dose-range finding study with two tester strains is reported as a part of the mutation assay. In the second part of this experiment, the test material was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2 °C. The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (109 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test material in Milli-Q water and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vvortex mixer and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted. An additional direct plate assay was performed with the tester strains TA100, WP2uvrA and TA1535.
In compliance with the OECD guideline No 471, there is no requirement for verification of a clear positive response. Since the test results of the mutation experiment showed clear positive responses, the follow-up experiment (pre-incubation assay) was not performed.

NUMBER OF REPLICATIONS: Testing was performed in triplicate

COLONY COUNTING
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually. Evidence of test material precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
The assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at the testing facility.
b) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5 % of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

DATA EVALUATION
- In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
- A test material is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
- A test material is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535 & TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

In tester strain TA100 and TA1535, in the presence of S9-mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in the tester strains TA1537, TA98 and TA1537, TA98 and TA100 in the absence of S9-mix and in tester strains TA1537, TA98 and WP2uvrA in the presence of S9-mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The test material was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

DIRECT PLATE ASSAY
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period in any tester strain.
- Toxicity: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
- Cytotoxicity, as evidenced by a reduction of the bacterial background lawn, was observed in the tester strains TA1537, TA98 and TA100 in the absence of S9-mix and in tester strains TA1537, TA98 and WP2uvrA in the presence of S9-mix.
Mutagenicity:
- In tester strain WP2uvrA, the test material induced an up to 2.2-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the historical control data range and was not dose-related.
- In tester strain TA100, the test material induced up to 3.0-fold dose-related increases in the number of revertant colonies in the presence of S9-mix. The increases observed were above the laboratory historical control data ranges and more than two-fold the concurrent solvent control. In the absence of S9-mix, the test item induced dose-related increases. However, the increases were only 1.6-fold and within the historical control data range.
- In tester strain TA1535, the test material induced an up to 3.3-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was above the laboratory historical control data ranges and more than three-fold the concurrent solvent control.

ADDITIONAL DIRECT PLATE ASSAY
To verify the mutagenic responses observed in the mutation assay, an additional experiment was performed. In this additional mutation experiment the test item was tested at a concentration range of 52 to 5000 µg/plate in tester strain TA100 in the absence of S9-mix and the tester strains TA100, WP2uvrA and TA1535 in the presence of S9-mix.
- Precipitate: Precipitation of the test material on the plates was not observed at the start or at the end of the incubation period.
- Toxicity: Cytotoxicity, as evidenced by a reduction in the bacterial background lawn and the presence of microcolonies, was observed in the tester strains TA100 and WP2uvrA in the presence of S9-mix.
Mutagenicity:
-In tester strain TA100, the test material induced up to 1.9-fold dose-related increases in the number of revertant colonies in the presence of S9-mix. The increases observed were above the laboratory historical control data ranges but less than 2-fold the concurrent solvent control. In the absence of S9-mix, the test material induced dose-related increases, however, the increases were only 1.5-fold and within the historical control data range.
- In tester strain TA1535, the test material induced an up to 2.5-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was above the laboratory historical control data ranges but less than 3-fold the concurrent solvent control.


DISCUSSION
- The negative control values were within the laboratory historical control data ranges.
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA1537 (absence of S9-mix in the first experiment). The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 200 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.
- In the absence of S9-mix, no biologically relevant increases in the number of revertant colonies were observed.
- In the presence of S9-mix, the test material induced increases in the number of revertant colonies in the tester strains TA1535, TA100 and WP2uvrA. The increases were reproducible in the tester strains TA1535 and TA100. The increases were above the laboratory historical control data range in both the initial and the additional mutation experiment, but were only more than two- and three-fold in the initial experiment.
- Taken together, since 3.0- to 3.3-fold increases were observed in the presence of S9-mix in the tester strains TA100 and TA1535, respectively, and the results were reproducible, these increases are considered biologically relevant and the test item is mutagenic

Any other information on results incl. tables

Dose-Range Finding Test: Mutagenic Response of the test material in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one Salmonella typhimurium and one Escherichia coli strain.
	TA100					WP2uvrA
Without S9-mix
Positive control	748	±	13		1243		±	80
Solvent control	81	±	18		37		±	5
1.7	84	±	12		32		±	7
5.4	80	±	14		41		±	2
17	91	±	12		33		±	2
52	88	±	16		32		±	4
164	102	±	29		41		±	5
512	114	±	21		31		±	10
1600	129	±	20	^n	33		±	6
5000	99	±	58	^s m NP	23		±	4	^n NP
With S9-mix
Positive control	1251	±	39		439		±	44
Solvent control	71	±	7		24		±	7
1.7	91	±	4		53		±	13
5.4	79	±	12		38		±	5
17	87	±	3		42		±	7
52	73	±	16		33		±	12
164	108	±	9		35		±	8
512	136	±	16		52		±	15
1600	184	±	15		50		±	9
5000	212	±	18	^n NP	23		±	7	^n m NP

 

NP	No precipitate
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Mutation Experiment: Mutagenic Response of the test material in the Salmonella typhimurium Reverse Mutation Assay 

Dose (µg/plate)		Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.
		TA1535					TA1537					TA98
Without S9-mix
Positive control	1057		±	31		1407		±	85		1231		±	150
Solvent control	14		±	2		6		±	2		14		±	3
52	14		±	4		8		±	2		16		±	3
164	11		±	2		6		±	4		12		±	2
512	12		±	6		6		±	2		14		±	2
1600	13		±	6		3		±	2	^n	18		±	2	^n
5000	16		±	4	^n NP	5		±	8	^m NP	19		±	9	^s NP
With S9-mix
Positive control	330		±	26		399		±	72		1604		±	122
Solvent control	10		±	4		7		±	3		24		±	9
52	16		±	8		8		±	1		24		±	8
164	19		±	8		10		±	2		19		±	4
512	27		±	3		7		±	1		27		±	8
1600	33		±	10		5		±	1	^n	30		±	13	^n
5000	23		±	6	^n NP	7		±	3	^s NP	21		±	4	^s NP

 

NP	No precipitate
m	Bacterial background lawn moderately reduced
n	Normal bacterial background lawn
s	Bacterial background lawn slightly reduced

 

Additional Mutation Experiment: Mutagenic Response of the test material in the Salmonella typhimurium Reverse Mutation Assay and in the Escherichia coli Reverse Mutation Assay 

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain.
	TA1535					TA100					WP2uvrA
Without S9-mix
Positive control					919		±	60
Solvent control					109		±	11
52					121		±	7
164					118		±	12
512					127		±	15
1600					155		±	4
2500					163		±	21
5000					104		±	41	^n NP
With S9-mix
Positive control	439	±	12		2379		±	41		514		±	6
Solvent control	13	±	2		124		±	12		44		±	19
52	13	±	6		125		±	11		38		±	10
164	11	±	3		134		±	16		47		±	2
512	14	±	6		179		±	8		56		±	12
1600	26	±	6		236		±	22		55		±	12
2500	32	±	1		235		±	34	^n	56		±	5	^n
5000	19	±	6	^n NP					^e NP MC					^e NP MC

 

MC	Microcolonies
NP	No precipitate
e	Bacterial background lawn extremely reduced
n	Normal bacterial background lawn

  

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, it was concluded that the test material was mutagenic.

Executive summary:

The genetic toxicity of the test material was investigated in accordance with the standardised guidelines OECD 471 and EU Method B13/14, under GLP conditions.

The objective of this study was to determine the potential of the test material and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The test material was dissolved in Milli-Q water.  

In the dose range finding study, the test material was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction in the bacterial background lawn, was observed in tester strain TA100 in the absence of S9-mix and in strain WP2uvrA in the presence of S9-mix.

In the first mutation experiment, the test material was tested up to concentrations of 5000 µg/plate in the strains TA1535, TA1537 and TA98. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction in the bacterial background lawn, was observed in the tester strains TA1537 and TA98 in the absence and presence of S9-mix.

In tester strain WP2uvrA, the test material induced an up to 2.2-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was within the historical control data range and was not dose-related.

In tester strain TA100, the test material induced up to 3.0-fold dose-related increases in the number of revertant colonies in the presence of S9-mix. The increases observed were above the laboratory historical control data ranges and more than two-fold the concurrent solvent control. In the absence of S9-mix, the test item induced dose-related increases. However, the increases were only 1.6-fold and within the historical control data range.

In tester strain TA1535, the test material induced an up to 3.3-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was above the laboratory historical control data ranges and more than three-fold the concurrent solvent control.

To verify the mutagenic responses observed in the first mutation experiment, an additional experiment was performed. In the additional experiment the test item was tested at a concentration range of 52 to 5000 μg/plate in tester strain TA100 in the absence of S9-mix and the tester strains TA100, WP2uvrA and TA1535 in the presence of S9-mix. The test material did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a reduction in the bacterial background lawn and the presence of microcolonies, was observed in the tester strains TA100 and WP2uvrA in the presence of S9-mix.

In tester strain TA100, the test material induced up to 1.9-fold dose-related increases in the number of revertant colonies in the presence of S9-mix. The increases observed were above the laboratory historical control data ranges.

In tester strain TA1535, the test material induced an up to 2.5-fold increase in the number of revertant colonies in the presence of S9-mix. The increase observed was above the laboratory historical control data ranges. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

In the absence of S9-mix, no biologically relevant increases in the number of revertant colonies were observed.

In the presence of S9-mix, the test material induced increases in the number of revertant colonies in the tester strains TA1535, TA100 and WP2uvrA. The increases were reproducible in the tester strains TA1535 and TA100. The increases were above the laboratory historical control data range in both the initial and the additional mutation experiment, but were only more than two- and three-fold in the initial experiment.

Taken together, since 3.0- to 3.3-fold increases were observed in the presence of S9-mix in the tester strains TA100 and TA1535, respectively, and the results were reproducible, these increases are considered biologically relevant and the test material is mutagenic.

Under the conditions of this study, it was concluded that the test material was mutagenic.