Ammonium formate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study according to OECD guideline 471, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as Escherichia coli strain WP2 uvrA were incubated with Ammonium Formate in the following concentrations: 0, 5.0, 16, 50, 160, 500, 1600 and 5000 µg/plate for 48h in the presence and absence of metabolic activation (mammalian S9 -Mix) using the plate incorporation method for the initial test and the pre-incubation method for the conformatory test, result: negative

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:

2019-07-02 to 2019-11-28

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his locus

Species / strain / cell type:

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

Additional strain / cell type characteristics:

DNA polymerase A deficient

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: S9 Liver Mix
- source of S9 : rat; the S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
- concentration or volume of S9 mix and S9 in the final culture medium 10% S9 Mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) The Quality Control & Production Certificate of each lot of S9 was obtained from the supplier.

Test concentrations with justification for top dose:

Selection of the concentration range for the initial mutation test was done on the basis of solubility test and concentration range finding test (informatory toxicity test). The chosen, already investigated concentration levels were not modified for the confirmatory mutation test. Based on the information about the solubility properties of the test item, a stock solution with a nominal concentration of 50 mg/mL was prepared in ultrapure water (ASTM Type I), and further diluted in 6 steps by factor of approximately √10.
The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO; aqueous solvents (water)
- Justification for choice of solvent/vehicle: In the study two types of vehicle control were used depending on the solubility of the test item and the solubility of positive control reference items.

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine, NPD for TA98 (without metabolic activation; 4µg/plate in DMSO); with metabolic activation 2-aminoanthracene for all strains (2µg/plat for S. typhimurium, 50µg/plate for E.coli)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): about 1 to 3E+09 cells/mL
- Test substance added in medium; in agar (plate incorporation); preincubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 min at 37°C
- Exposure duration/duration of treatment: 48h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Background growth inhibition
- Any supplementary information relevant to cytotoxicity: No

Rationale for test conditions:

As recommended by OECD guideline 471.

Evaluation criteria:

A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
A test item is considered mutagenic if:
-a dose–related increase in the number of revertants occurs and/or;
-a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
-in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control (water),
-in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control (water).

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: Not reported
- Data on osmolality: Not reported
- Possibility of evaporation from medium: No
- Water solubility: No effects observed
- Precipitation and time of the determination: No precipitation observed
RANGE-FINDING/SCREENING STUDIES (if applicable): A range-finding study was conducted in tester strains TA98 and TA100.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The revertant colonies per plate were within the range of the acceptance criteria

Ames test:
- Signs of toxicity : No
- Individual plate counts : see 'Any other information on results incl. tables'
- Mean number of revertant colonies per plate and standard deviation . see 'Any other information on results incl. tables'


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: see 'Any other information on results incl. tables'
- Negative (solvent/vehicle) historical control data: see 'Any other information on results incl. tables'

Summary Table of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	13.3	0.85	35.7	0.84	82.0	1.02	100.0	1.20
DMSO Control	19.0	1.00	35.3	1.00	–	–	99.7	1.00
Ultrapure Water Control	15.7	1.00	42.7	1.00	80.3	1.00	83.0	1.00
5000	18.3	1.17	35.0	0.82	87.3	1.09	111.3	1.34
1600	19.0	1.21	35.0	0.82	89.0	1.11	113.7	1.37
500	23.0	1.47	32.7	0.77	84.3	1.05	111.0	1.34
160	20.0	1.28	28.3	0.66	89.7	1.12	105.7	1.27
50	15.3	0.98	34.0	0.80	78.7	0.98	106.0	1.28
16	19.3	1.23	34.3	0.80	81.7	1.02	107.0	1.29
5	22.7	1.45	34.3	0.80	80.3	1.00	103.7	1.25
NPD (4mg/plate)	574.7	30.25	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	817.3	10.17	–	–
2AA (2mg/plate)	–	–	797.3	22.57	–	–	928.0	9.31

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	14.7	0.98	23.3	0.82	62.7	0.99	73.0	0.96	11.7	1.30	13.7	1.14	6.3	0.90	9.3	1.17	32.0	1.17	37.3	0.85
DMSO Control	12.7	1.00	22.3	1.00	–	–	66.3	1.00	–	–	11.0	1.00	5.3	1.00	8.0	1.00	–	–	37.3	1.00
Ultrapure Water Control	15.0	1.00	28.3	1.00	63.0	1.00	76.0	1.00	9.0	1.00	12.0	1.00	7.0	1.00	8.0	1.00	27.3	1.00	44.0	1.00
5000	21.3	1.42	22.3	0.79	83.0	1.32	93.3	1.23	8.7	0.96	10.7	0.89	7.0	1.00	7.0	0.88	40.0	1.46	47.3	1.08
1600	20.7	1.38	20.7	0.73	76.0	1.21	91.3	1.20	10.7	1.19	11.0	0.92	6.3	0.90	6.3	0.79	38.0	1.39	39.3	0.89
500	23.7	1.58	18.3	0.65	65.3	1.04	91.7	1.21	9.0	1.00	11.7	0.97	6.0	0.86	7.7	0.96	32.0	1.17	35.0	0.80
160	18.7	1.24	17.0	0.60	63.7	1.01	91.3	1.20	12.7	1.41	12.3	1.03	4.7	0.67	7.3	0.92	30.0	1.10	28.0	0.64
50	15.3	1.02	25.3	0.89	74.3	1.18	83.0	1.09	13.3	1.48	11.3	0.94	6.0	0.86	7.3	0.92	28.7	1.05	29.3	0.67
16	21.0	1.40	24.7	0.87	69.3	1.10	103.7	1.36	12.0	1.33	10.3	0.86	4.3	0.62	10.3	1.29	28.3	1.04	34.3	0.78
NPD (4mg/plate)	417.3	32.95	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	837.3	13.29	–	–	992.0	110.22	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	569.3	106.75	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	688.0	25.17	–	–
2AA (2mg/plate)	–	–	1936.0	86.69	–	–	770.7	11.62	–	–	194.7	17.70	–	–	112.3	14.04	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	180.7	4.84

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichiacoli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated Control	12.7	0.83	23.0	0.91	80.7	1.19	90.3	1.10	13.3	1.25	13.3	1.11	5.7	0.94	8.0	0.89	25.0	0.94	35.3	0.86
DMSO Control	14.0	1.00	18.0	1.00	–	–	87.0	1.00	–	–	13.3	1.00	6.0	1.00	7.0	1.00	–	–	38.7	1.00
Ultrapure Water Control	15.3	1.00	25.3	1.00	68.0	1.00	82.3	1.00	10.7	1.00	12.0	1.00	6.0	1.00	9.0	1.00	26.7	1.00	41.0	1.00
5000	16.0	1.04	14.0	0.55	62.7	0.92	130.7	1.59	14.3	1.34	11.7	0.97	5.3	0.89	8.7	0.96	32.7	1.23	43.3	1.06
1600	21.7	1.41	23.7	0.93	69.7	1.02	116.7	1.42	10.0	0.94	10.7	0.89	4.3	0.72	7.0	0.78	39.7	1.49	57.0	1.39
500	21.0	1.37	16.7	0.66	73.3	1.08	112.3	1.36	8.3	0.78	9.7	0.81	7.7	1.28	8.0	0.89	31.3	1.18	39.0	0.95
160	19.3	1.26	20.0	0.79	67.0	0.99	97.0	1.18	10.3	0.97	9.7	0.81	4.7	0.78	7.3	0.81	31.7	1.19	39.3	0.96
50	14.0	0.91	20.0	0.79	68.7	1.01	107.3	1.30	10.3	0.97	10.0	0.83	7.0	1.17	7.3	0.81	27.0	1.01	39.3	0.96
16	17.7	1.15	20.3	0.80	69.3	1.02	95.3	1.16	8.0	0.75	9.3	0.78	7.0	1.17	9.0	1.00	25.0	0.94	39.3	0.96
NPD (4mg/plate)	460.0	32.86	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	1072.0	15.76	–	–	1064.0	99.75	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	355.3	59.22	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	893.3	33.50	–	–
2AA (2mg/plate)	–	–	1056.0	58.67	–	–	770.7	8.86	–	–	190.3	14.28	–	–	87.7	12.52	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	197.0	5.09

Historical Control Values for Revertants/Plate (for the Period of 2016-2018)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.9	88.7	10.7	7.9	28.0
		SD	2.0	10.5	0.6	0.7	3.7
		Minimum	8	62	4	2	12
		Maximum	36	128	21	18	50
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	24.0	106.5	11.2	8.3	35.1
		SD	1.7	10.5	0.3	0.7	3.9
		Minimum	11	71	3	2	16
		Maximum	40	152	20	17	59
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	17.5	80.6	10.7	7.5	25.9
		SD	1.2	10.6	0.8	0.7	2.6
		Minimum	9	58	4	2	12
		Maximum	35	124	21	18	52
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	22.3	95.6	10.9	8.1	34.5
		SD	1.1	10.3	0.6	0.7	4.2
		Minimum	10	65	3	2	16
		Maximum	37	146	24	19	58
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	18.6	87.0	11.1	7.9	29.8
		SD	1.9	11.5	0.8	0.5	4.7
		Minimum	10	60	3	2	13
		Maximum	31	120	24	17	55
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	23.6	105.0	11.3	8.1	36.8
		SD	1.8	11.7	0.7	0.6	4.0
		Minimum	13	76	5	3	18
		Maximum	41	148	19	17	63
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	364.6	1095.3	987.7	564.4	911.6
		SD	111.2	197.3	119.3	73.8	112.8
		Minimum	182	543	409	137	361
		Maximum	844	2200	2123	2265	1995
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1419.2	1580.8	168.2	148.2	203.3
		SD	249.7	337.3	22.0	16.3	5.3
		Minimum	281	688	93	71	132
		Maximum	3421	3333	349	367	364

Conclusions:

The test item Ammonium formate was tested with regard to its potential mutagenic activity using the Bacterial Reverse Mutation Assay. No biologically relevant increases were observed in the number of revertant colonies in any of the five tester strains following treatment with Ammonium Formate at any concentration levels, either in the presence or absence of S9 Mix in the performed experiments.
Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Ammonium formate has no mutagenic activity in the bacterial tester strains under the test conditions used in this study.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA97a, TA98, TA100, TA102, TA1535 and the Escherichia coli strain WP2 uvrA were exposed to Ammonium Formate dissolved in ultrapure water in concentrations of 0 (control), 5.0, 16, 50, 160, 500, 1600, and 5000 µg/plate in all strains in the absence and presence of mammalian metabolic activation (rat liver S9 mix). The assay was performed using the plate incorporation method for the initial mutation test and the pre-incubation method for the confirmatory mutation test.

The test substance was tested up to the maximum concentration of 5000 µg/plate as recommended by the guideline. No cytotoxic effects were noted in all strains in the presence and absence of metabolic activation. The positive controls induced the appropriate responses (more than 3-fold increase of revertants) in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

No biologically relevant increases were observed in the number of revertant colonies in any of the five tester strains following treatment with Ammonium Formate at any concentration levels, either in the presence or absence of S9 Mix in the performed experiments. Based on the results obtained under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item Ammonium Formate has no mutagenic activity in the bacterial tester strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results obtained from the bacterial reverse mutation test conducted according to OECD guideline 471, Ammonium Formate showed no significant increase of revertants over background and thus, does not need to be classified as mutagenic according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).