Hydroxylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

/ AT reversion site not covered, no historical control data provided, no concentrations of positive controls provided, no data on dose-finding study

Data source

Reference

Reference Type:

publication

Title:

Salmonella Mutagenicity Tests: V. Results from the Testing of 311 Chemicals.

Author:

Zeiger E et al.

Year:

1992

Bibliographic source:

Environmental and Molecular Mutagenesis 19 (21): 2 - 141

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

/ AT reversion site not covered, no historical control data provided, no concentrations of positive controls provided, no data on dose-finding study

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxylammonium chloride

EC Number:

226-798-2

EC Name:

Hydroxylammonium chloride

Cas Number:

5470-11-1

Molecular formula:

ClH.H3NO

IUPAC Name:

hydroxyammonium chloride

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats and male hamsters, treated with Aroclor 1254.

Test concentrations with justification for top dose:

main experiment:
TA 97: 33, 100, 251, 333, 667 µg/plate (- S9 mix, + 10% S9 mix (hamster/rat)); 33, 100, 200, 333, 667 µg/plate (+ 30% S9 mix (hamster/rat))
TA 98: 33, 100, 251, 333, 667 µg/plate (- S9 mix, + 10% S9 mix (10%, hamster/rat)); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat))
TA 1535: 33, 100, 200, 333, 667 µg/plate (- S9 mix, + 30% S9 mix (hamster/rat)); 33, 100, 251, 333, 667 µg/plate (+ 10% S9 mix (hamster)); 33, 100, 250, 333, 667 µg/plate (+ 10% S9 mix (rat))

TA 100:
test 1: 10, 33, 100, 333, 667 µg/plate (-S9 mix) ; 100, 333, 500, 750, 1000 µg/plate (+ 10% S9 mix (hamster); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat) and + 10% S9 mix (rat))
test 2: 10, 33, 100, 333, 667 µg/plate (-S9 mix) ; 33, 100, 251, 333, 667 µg/plate (+ 10% S9 mix (hamster); 33, 100, 200, 333, 667 µg/plate (+ 10% S9 mix (rat)); 33, 100, 333, 1000, 2000 µg/plate (+ 30% S9 mix (hamster/rat))
test 3: 100, 333, 500, 750, 1000 µg/plate (+ 30% S9 mix (hamster) and + 10% S9 mix (rat)); 33, 100, 200, 333, 667 µg/plate (+ 30% S9 mix (rat))
test 4: 33, 100, 251, 333, 667 µg/plate (+ 30% S9 mix (hamster) + 10% S9 mix (rat)); 100, 333, 500, 750, 1000 µg/plate (+ 30% S9 mix (rat))
test 5: 33, 100, 251, 333, 667 µg/plate (+ 30% S9 (rat))

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine (4-NPD), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: The main assay was carried out in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number his+ colonies and/or clearing of the bacterial background lawn

Rationale for test conditions:

Reference to "Zeiger E, Drake JW (1980): An environmental mutagenesis test development programme. In Montesano R, Bartsch H, Tomatis L (eds):
“Molecular and Cellular Aspects of Carcinogen Screening Tests." Lyon: IARC Scientific Publications, No. 27, pp 303-313."

Evaluation criteria:

The test article is judged to be mutagenic or weakly mutagenic, if it produced a reproducible dose-related increase in his+ revertants compared to the corresponding solvent control under a single metabolic activation condition, in replicate trials.

Statistics:

Mean values and standard error of means were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased to less than half compared to solvent control starting at 333 μg/ plate without S9 mix (52%) and at 667 μg/ plate with 10% S9 mix (hamster, 51%)

Vehicle controls validity:

other: no historical control data given

Untreated negative controls validity:

not applicable

Positive controls validity:

other: no historical control data given

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased to less than half compared to solvent control starting at 1000 μg/ plate with 30% S9 mix (hamster, 100%)

Vehicle controls validity:

other: no historical control data given

Untreated negative controls validity:

not applicable

Positive controls validity:

other: no historical control data given

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased to less than half compared to solvent control in exp.1 starting at 750 μg/plate with 10% S9 mix (hamster, 100%) and in exp.1 and 2 at 2000 μg/plate with 30% S9 mix (hamster, 63% and 55% respectively)

Vehicle controls validity:

other: no historical control data given

Untreated negative controls validity:

not applicable

Positive controls validity:

other: no historical control data given

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

number of revertant colonies decreased to less than half compared to solvent control starting at 333 μg/plate without S9 mix (59%)

Vehicle controls validity:

other: no historical control data given

Untreated negative controls validity:

not applicable

Positive controls validity:

other: no historical control data given

Remarks on result:

other: Cytotoxicity was furthermore detected in exp.3 at 1000 μg/plate with 30% S9 mix (hamster, 85%) and starting at 750 μg/plate with 10% S9 mix (rat, 89%), in exp. 4 it started at 750 μg/plate with 30% S9 mix (rat, 85%)

Any other information on results incl. tables

Table 1: Summary of test results (main assay)

With or without S9 -mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates)
		Frameshift type		Base-pair substitution type
		TA97	TA98	TA100					TA1535
				exp. 1	exp. 2	exp. 3	exp. 4	exp. 5
– S9	Solvent control (water)	86 ± 7.8	28 ± 4.3	118 ± 6.6	160 ± 11.3	-			27 ± 2.1
	10	-		103 ± 3.8	147 ± 11.6				-
	33	105 ± 5.8	23 ± 0.0	102 ± 5.0	138 ± 3.3				28 ± 3.2
	100	82 ± 7.4	24 ± 2.8	100 ± 3.7	174 ± 3.1				22 ± 3.6
	200	-							17 ± 2.0
	250								-
	251	64 ± 5.5	24 ± 0.3	-
	333	41 ± 4.0 s	20 ± 1.7	126 ± 2.6	173 ± 4.9				11 ± 1.8
	500	-	-	-	-				-
	667	18 ± 1.5	21 ± 0.0	64 ± 3.2	94 ± 10.8				9 ± 2.0
	750	-							-
	1000
	2000
	Positive controls	9AA	4-NPD	SAZ					SAZ
	Mean No. of colonies/plate (average of 3 plates)	692 ± 11.7	582 ± 21.3	440 ± 3.1	570 ± 42.9	-			257 ± 33.5
+ 10% S9 (hamster)	Solvent control (water)	208 ± 7.5	32 ± 2.6	118 ± 7.3	124 ± 2.3	-			15 ± 2.4
	10	-							-
	33	216 ± 6.0	29 ± 1.5	-	110 ± 4.2				16 ± 1.5
	100	207 ± 6.9	26 ± 2.9	117 ± 6.4	120 ± 1.3				14 ± 1.5
	200	-							-
	250
	251	197 ± 5.2	26 ± 0.9	-	151 ± 8.4				21 ± 1.0
	333	156 ± 1.8	25 ± 3.2	139 ± 3.3	155 ± 0.6				19 ± 2.7
	500	-		62 ± 2.3	-				-
	667	101 ± 11.0	29 ± 3.0	-	198 ± 6.1				24 ± 0.9
	750	-		0 ± 0 s	-				-
	1000			0 ± 0 s
	2000			-
	Positive control	2AA
	Mean No. of colonies/plate (average of 3 plates)	646 ± 8.1	244 ± 12.5	440 ± 12.5	383 ± 16.7	-			111 ± 6.7
+ 30% S9 (hamster)	Solvent control (water)	161 ± 9.4	31 ± 0.9	123 ± 5.8	132 ± 4.8	132 ± 5.8	176 ± 18.0	-	12 ± 1.3
	10	-							-
	33	183 ± 9.0	41 ± 1.5	126 ± 7.0	164 ± 4.1	-	141 ± 5.4		17 ± 0.9
	100	169 ± 7.4	31 ± 2.0	100 ± 2.6	158 ± 3.5	132 ± 1.9	115 ± 7.4		11 ± 0.9
	200	154 ± 23.8	-				-		13 ± 1.5
	250	-							-
	251						146 ± 9.9
	333	138 ± 18.2	27 ± 7.6 s	166 ± 4.6	210 ± 3.5	216 ± 8.0	158 ± 9.5		15 ± 0.9
	500	-	-			201 ± 6.9	-		-
	667	105 ± 11.1				-	214 ± 11.9		14 ± 2.3
	750	-				91 ± 7.1	-		-
	1000		0 ± 0.0 s	104 ± 7.4	130 ± 7.8 s	20 ± 6.3 s
	2000		t	45 ± 12.3 s	60 ± 3.2 s	-
	Positive control	2AA
	Mean No. of colonies/plate (average of 3 plates)	898 ± 51.3	171 ± 13.5	856 ± 40.5	748 ± 11.3	578 ± 14.0	503 ± 13.5	-	130 ± 7.6
+ 10% S9 (rat)	Solvent control (water)	145 ± 6.0	50 ± 2.7	126 ± 10.5	121 ± 1.8	132 ± 4.2	136 ± 12.1	-	12 ± 1.5
	10	-
	33	136 ± 6.2	51 ± 4.6	128 ± 7.1	148 ± 9.2	-	156 ± 6.7	-	15 ± 1.5
	100	136 ± 6.6	49 ± 2.3	169 ± 7.8	151 ± 3.6	122 ± 4.2	162 ± 8.8		19 ± 3.0
	200	-		-	182 ± 4.5	-	-		-
	250				-				17 ± 4.1
	251	159 ± 6.1	46 ± 5.5				193 ± 13.7		-
	333	156 ± 4.0	54 ± 5.9	188 ± 3.5	206 ± 5.9	170 ± 7.7	195 ± 11.0		17 ± 3.1
	500	-		-	-	105 ± 3.8	-		-
	667	84 ± 2.6	55 ± 7.4		185 ± 8.5	-	229 ± 13.5		15 ± 2.3
	750	-			-	14 ± 2.3	-		-
	1000			142 ± 9.6		0 ± 0.0 s
	2000			74 ± 3.2 s		-
	Positive control	2AA
	Mean No. of colonies/plate (average of 3 plates)	1421 ± 8.7	118 ± 9.5	1215 ± 17.1	854 ± 155.7	1582 ± 29.0	650 ± 64.0	-	302 ± 7.3
+ 30% S9 (rat)	Solvent control (water)	139 ± 19.7	34 ± 3.3	110 ± 3.0	190 ± 4.3	161 ± 3.7	138 ± 5.7	159 ± 3.7	13 ± 0.9
	10	-
	33	130 ± 7.5	33 ± 1.9	117 ± 6.7	187 ± 11.4	164 ± 4.3	-	161 ± 4.5	12 ± 3.5
	100	138 ± 3.5	32 ± 0.9	130 ± 3.5	191 ± 6.0	125 ± 5.5	143 ± 8.5	174 ± 6.2	18 ± 1.5
	200	96 ± 4.8	-			143 ± 9.0	-	-	18 ± 0.9
	250	-							-
	251							165 ± 1.7
	333	99 ± 11.5	36 ± 3.2	172 ± 4.9	184 ± 5.9	175 ± 7.0	162 ± 5.5	178 ± 6.4	18 ± 1.2
	500	-	-			-	143 ± 2.8	-	-
	667	69 ± 4.2				173 ± 12.1	-	242 ± 20.5	12 ± 2.6
	750	-				-	21 ± 0.3	-
	1000		28 ± 1.3	211 ± 13.3	225 ± 18.1		0 ± 0.0 s
	2000		18 ± 3.5 s	66 ± 3.1 s	86 ± 3.1 s		-
	Positive control	2AA
	Mean No. of colonies/plate (average of 3 plates)	537 ± 34.9	499 ± 10.4	1536 ± 1.3	673 ± 11.6	1536 ± 16.6	1525 ± 23.7	1161 ± 19.9	71 ± 10.3

 2AA = 2-aminoanthracene

4-NPD = 4-nitro-o-phenylenediamine

SAZ = sodium azide

9AA = 9-aminoacridine

s = slight clearing of background lawn

t = complete clearing of background lawn