Hydrogen iodide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22.05.2019 - 14.10.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
To establish a dose response effect at least six dose levels with adequately spaced concentrations were tested. The maximum dose level was 5000 µg/plate.

Vehicle / solvent:

- Solvent used: deionised tap water
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD - without S9 mix: TA1537, TA 98 2-aminoanthracene, 2-AA - with S9 mix: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3

METHOD OF TREATMENT/ EXPOSURE:
- Test item preparation: On the day of the experiment, the test item was dissolved in deionised tap water. The test item was neutralised with NaOH2N. The pH value of the stock solution was 7 after neutralisation.

A pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I since the acceptance criteria were met.

Experiment I (Plate Incorporation)
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension,
2000 μL Overlay agar

Experiment II (Pre-Incubation)
The following materials were mixed in a test tube and incubated at 37 °C for 60 minutes.
100 μL Test solution at each dose level (solvent or reference mutagen solution (positive control)),
500 μL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 μL Bacteria suspension.
After pre-incubation 2.0 mL overlay agar (45 °C) was added to each tube.

The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
In parallel to each test a sterile control of the test item was performed. Therefore, 100 μL of the stock solution, 500 μL S9 mix / S9 mix substitution buffer were mixed with 2.0 mL overlay agar and poured on minimal agar plates.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Evident as a reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2500 µg/plate without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item occurred up to the highest investigated dose. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	5000	5000
TA 1537	/	/	5000	/
TA 98	/	/	/	/
TA 100	/	/	5000	/
WP2 uvrA	/	/	2500 - 5000	/

/ = no toxic effects (induction factor ≥ 0.5)

 

Summary results from Experiment I:

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without activation	Deionised water		16 ± 2	13 ± 7	27 ± 5	125 ± 6	56 ± 13
	Untreated control		13 ± 5	12 ± 4	33 ± 8	130 ± 20	51 ± 16
	Test Item	3 µg	14 ± 7	12 ± 4	33 ± 6	129 ± 5	57 ± 6
		10 µg	15 ± 6	14 ± 4	35 ± 4	125 ± 13	62 ± 7
		33 µg	12 ± 3	18 ± 1	29 ± 3	138 ± 11	59 ± 6
		100 µg	11 ± 5	16 ± 3	35 ± 8	145 ± 7	60 ± 6
		333 µg	13 ± 4	13 ± 1	32 ± 7	135 ± 12	45 ± 3
		1000 µg	12 ± 5	16 ± 4	44 ± 7	140 ± 6	48 ± 8
		2500 µg	9 ± 5	10 ± 2	32 ± 9	126 ± 13	40 ± 5
		5000 µg	10 ± 5	13 ± 2	21 ± 5	105 ± 10	35 ± 11
	NaN3	10 µg	1163 ± 29			1952 ± 95
	4-NOPD	10 µg			295 ± 9
	4-NOPD	50 µg		69 ± 3
	MMS	2.0 µL					1050 ± 79
With activation	Deionised water		14 ± 5	13 ± 5	36 ± 1	125 ± 24	65 ± 11
	Untreated control		15 ± 6	16 ± 5	38 ± 6	138 ± 7	57 ± 4
	Test Item	3 µg	19 ± 2	16 ± 5	40 ± 11	131 ± 8	59 ± 7
		10 µg	15 ± 4	12 ± 1	43 ± 2	153 ± 23	65 ± 11
		33 µg	14 ± 4	12 ± 0	36 ± 9	147 ± 15	62 ± 6
		100 µg	13 ± 6	10 ± 4	32 ± 5	129 ± 4	59 ± 8
		333 µg	13 ± 4	11 ± 2	37 ± 4	129 ± 3	62 ± 3
		1000 µg	10 ± 3	15 ± 5	40 ± 2	159 ± 21	63 ± 8
		2500 µg	13 ± 3	12 ± 4	38 ± 7	143 ± 11	63 ± 10
		5000 µg	10 ± 4	10 ± 4	40 ± 8	131 ± 9	62 ± 6
	2-AA	2.5 µg	354 ± 55	405 ± 31	3308 ± 413	4604 ± 229
	2-AA	10.0 µg					383 ± 30

Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.

 

Summary results from Experiment II:

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98*	TA 100	WP2 uvrA
Without activation	Deionised water		14 ± 5	14 ± 4	26 ± 8	96 ± 8	44 ± 4
	Untreated control		9 ± 5	16 ± 5	32 ± 1	102 ± 9	46 ± 11
	Test Item	33 µg	13 ± 2	11 ± 5	32 ± 6	109 ± 8	38 ± 8
		100 µg	15 ± 4	15 ± 4	28 ± 2	104 ± 1	46 ± 9
		333 µg	10 ± 6	12 ± 3	28 ± 2	103 ± 19	50 ± 5
		1000 µg	7 ± 3	9 ± 2	27 ± 9	104 ± 5	29 ± 4
		2500 µg	10 ± 5	6 ± 1	28 ± 6	73 ± 11	12 ± 3
		5000 µg	1 ± 1	3 ± 2	21 ± 2	2 ± 2	1 ± 1
	NaN3	10 µg	1221 ± 59		594 ± 49	1770 ± 80
	4-NOPD	10 µg			574 ± 24
	4-NOPD	50 µg		76 ± 8
	MMS	2.0 µL					646 ± 24
With activation	DMSO (Solvent control)		16 ± 2	13 ± 5	38 ± 3	100 ± 19	58 ± 6
	Untreated control		18 ± 4	17 ± 4	26 ± 6	95 ± 5	51 ± 4
	Test Item	33 µg	12 ± 3	12 ± 6	38 ± 12	101 ± 14	61 ± 6
		100 µg	15 ± 5	16 ± 1	34 ± 7	98 ± 2	61 ± 6
		333 µg	17 ± 6	12 ± 8	43 ± 3	100 ± 8	53 ± 6
		1000 µg	15 ± 6	12 ± 2	28 ± 7	96 ± 14	49 ± 3
		2500 µg	12 ± 3	10 ± 4	36 ± 12	89 ± 10	54 ± 6
		5000 µg	6 ± 1	10 ± 3	25 ± 8	83 ± 20	52 ± 6
	2-AA	2.5 µg	388 ± 55	295 ± 16	2449 ± 66	2825 ± 3
	2-AA	10.0 µg					370 ± 20

Positive Controls: NaN3 – sodium azide, 2-AA – 2-aminoanthracene, 4-NOPD – 4-nitro-o-phenylene-diamine, MMS – methyl methane sulfonate.

*Due to irregular bacteria growth this part was repeated and is reported as part of experiment II

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred in the overlay agar in the test tubes. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains only in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471

A study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The test was conducted according to OECD 471 (1997) and EU Method B.13/14 (30 May 2008). The assay was performed in three independent experiments. Experiment I and II were performed with and without liver microsomal activation (S9 mix). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

No precipitation of the test item occurred in the overlay agar in the test tubes. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in nearly all strains only in experiment II. No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.