Mercaptoacetic 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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ammonium mercaptoacetate

EC Number:

226-540-9

EC Name:

Ammonium mercaptoacetate

Cas Number:

5421-46-5

Molecular formula:

C2H4O2S.H3N

IUPAC Name:

ammonium sulfanylacetate

Details on test material:

Ammonium Thioglycolate 71%
Source: Bruno Bock Chemische Fabrik GmbH & Co KG
Description: colourless liquid
Batch number: 4804
Content of active ingredient: 71.2%

Method

Target gene:

Histidine reversion

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (from livers of male Sprague-Dawley rats  treated by Aroclor 1254)

Test concentrations with justification for top dose:

15, 50, 150, 500, 1500 and 5000 µg a.i./plate.
Two distinct experiments were performed using these doses.

Vehicle / solvent:

sterile distilled water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene, 1,8-Dihydroxyanthraquinone

Details on test system and experimental conditions:

METHOD DETAILS:
- Test concentrations:
. Preliminary study (on TA100): 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
. Main study: see the "Test concentration" field
- Number of replicates: 3
- Positive controls:
without S9-mix
. for TA1535 and TA100: N-ethyl-N'-nitro-N-nitrosoguanidine (5 and 2  µg/plate respectively)
. for TA1537: 9-aminoacridine (80 µg/plate)
. for TA98: 4-Nitroquinoline-1-oxide (0.2 µg/plate)
. for TA102: mitomycin C (0.5 µg/plate)
with S9-mix
. for TA1535, TA1357 and TA100: 2-aminoanthracene (2; 2 and 1µg/plate  respectively)
. for TA98: benzo(a)pyrene (5 µg/plate)
. for TA102: 1,8-Dihydroxyanthraquinone (10 µg/plate)
- Pre-incubation time: no
- Pre-incubation temperature: no
- Incubation time: 48h
- Incubation temperature: 37°C

EXAMINATION:
- Bacterial toxicity: determined by examination of background lawn growth
- Number of revertants / plate

ANALYTICAL DEVICE:  Colonies were counted electronically using a Domino Colony Counter.

Evaluation criteria:

The reverse mutation assay may be considered valid if the following  criteria are met: All tester strain cultures exhibit a characteristic number of spontaneous  revertants per plate in the vehicle and untreated controls. Acceptable  ranges are presented below with historical control ranges for 2001 and  2002 presented in Table 1.

Spontaneous Mutation Ranges:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
TA102 180 to 400

The test material may be considered positive in this test system if the  following criteria are met: The test material should have induced a reproducible, dose-related and  statistically significant  increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium 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:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

= 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

not examined

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY:
The test substance was toxic at 5000 µg/plate in the TA100 strain (without S9 mix: very weak bacterial background lawn, with S9 mix: sparse bacterial background lawn).

GENOTOXICITY:
No significant increases in the number of revertants were observed at any dose level, with or without metabolic activation.

Any other information on results incl. tables

Table 1:Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
136		9		351		15		5
139	(135)	14	(10)	350	(358)	18	(15)	5	(6)
129		7		373		13		8

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		TA102		TA98		TA1537
62		18		267		12		6
97	(73)	11	(14)	318	(301)	12	(12)	5	(5)
60		13		318		12		5

Table 2: Test Results: Experiment 1 – Without Metabolic Activation

Test Period		From : 25 March 2003						To : 28 March 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		TA102			TA98		TA1537
-	0	153 121 131	(135) 16.4#	9 7 8	(8) 1.0	367 310 385	(354) 39.2		13 16 24	(18) 5.7	6 7 11	(8) 2.6
-	15	128 114 130	(124) 8.7	6 9 5	(7) 2.1	353 296 434	(361) 69.3		16 17 17	(17) 0.6	5 5 3	(4) 1.2
-	50	104 129 118	(117) 12.5	12 12 5	(10) 4.0	399 360 399	(386) 22.5		16 14 12	(14) 2.0	5 6 4	(5) 1.0
-	150	144 113 118	(125) 16.6	11 8 8	(9) 1.7	406 372 360	(379) 23.9		15 7 16	(13) 4.9	4 5 6	(5) 1.0
-	500	101 106 118	(108) 8.7	11 13 11	(12) 1.2	362 359 393	(371) 18.8		15 8 12	(12) 3.5	5 3 3	(4) 1.2
-	1500	116 104 106	(109) 6.4	5 3 11	(6) 4.2	382 356 368	(369) 13.0		17 15 18	(17) 1.5	7 7 4	(6) 1.7
-	5000	0 V 0 V 0 V	(0) 0.0	3 S 9 S 5 S	(6) 3.1	204 221 153	(193) 35.4		17 S 14 S 13 S	(15) 2.1	0 S 0 S 4 S	(1) 2.3
Positive controls S9-Mix -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		MMC			4NQO		9AA
		3		5		0.5			0.2		80
		553 474 505	(511) 39.8	287 284 256	(276) 17.1	862 859 903	(875) 24.6		75 81 80	(79) 3.2	2605 2689 2688	(2661) 48.2

Table 3: Test Results: Experiment 1 – With Metabolic Activation

Test Period		From : 25 March 2003						To : 28 March 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		TA102			TA98		TA1537
+	0	173 145 180	(166) 18.5#	16 11 15	(14) 2.6	380 373 370	(374) 5.1		21 26 28	(25) 3.6	5 6 7	(6) 1.0
+	15	150 125 163	(146) 19.3	17 14 12	(14) 2.5	361 352 376	(363) 12.1		24 21 25	(23) 2.1	5 7 12	(8) 3.6
+	50	156 158 149	(154) 4.7	9 6 12	(9) 3.0	332 362 403	(366) 35.6		22 22 19	(21) 1.7	5 6 5	(5) 0.6
+	150	127 140 150	(139) 11.5	13 19 17	(16) 3.1	419 369 363	(384) 30.7		33 20 18	(24) 8.1	9 3 6	(6) 3.0
+	500	119 136 164	(140) 22.7	17 15 18	(17) 1.5	389 337 345	(357) 28.0		19 24 20	(21) 2.6	12 3 6	(7) 4.6
+	1500	133 130 121	(128) 6.2	8 6 9	(8) 1.5	404 412 304	(373) 60.2		23 25 19	(22) 3.1	3 14 2	(6) 6.7
+	5000	98 82 77	(86) 11.0	6 6 7	(6) 0.6	75 88 94	(86) 9.7		7 25 16	(16) 9.0	0 0 0	(0) 0.0
Positive controls S9-Mix +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		DAN			BP		2AA
		1		2		10			5		2
		1982 2219 1854	(2018) 185.2	378 376 401	(385) 13.9	789 787 779	(785) 5.3		220 262 259	(247) 23.4	284 336 315	(312) 26.2

Table 4: Test Results: Experiment 2 – Without Metabolic Activation

Test Period		From : 11 April 2003						To : 14 April 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		TA102			TA98		TA1537
-	0	95 102 128	(108) 17.4#	17 11 16	(15) 3.2	345 350 358	(351) 6.6		19 16 14	(16) 2.5	14 6 C	(10) 5.7
-	15	66 50 63	(60) 8.5	4 19 11	(11) 7.5	364 322 393	(360) 35.7		16 C 16	(16) 0.0	4 7 6	(6) 1.5
-	50	63 66 55	(61) 5.7	4 11 7	(7) 3.5	391 383 362	(379) 15.0		16 18 17	(17) 1.0	5 9 1	(5) 4.0
-	150	84 82 66	(77) 9.9	13 8 11	(11) 2.5	380 345 398	(374) 27.0		13 14 9	(12) 2.6	9 7 4	(7) 2.5
-	500	82 92 95	(90) 6.8	8 17 14	(13) 4.6	376 326 397	(366) 36.5		15 12 15	(14) 1.7	7 6 5	(6) 1.0
-	1500	86 103 85	(91) 10.1	11 8 6	(8) 2.5	326 390 329	(348) 36.1		11 13 22	(15) 5.9	9 6 3	(6) 3.0
-	5000	0 S 0 S 0 S	(0) 0.0	7 S 3 S 0 S	(3) 3.5	22 23 25	(23) 1.5		0 S 0 S 0 S	(0) 0.0	0 S 0 S XS	(0) 0.0
Positive controls S9-Mix -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		MMC			4NQO		9AA
		3		5		0.5			0.2		80
		419 381 274	(358) 75.2	305 227 222	(251) 46.5	1080 1040 C	(1060) 28.3		160 170 151	(160) 9.5	1379 1196 1552	(1376) 178.0

Table 5: Test Results: Experiment 2 – With Metabolic Activation

Test Period		From : 11 April 2003						To : 14 April 2003
With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		Base-pair substitution type							Frameshift type
		TA100		TA1535		TA102			TA98		TA1537
+	0	94 113 115	(107) 11.6#	11 8 11	(10) 1.7	397 326 384	(369) 37.8		22 26 24	(24) 2.0	5 7 7	(6) 1.2
+	15	135 92 107	(111) 21.8	5 11 14	(10) 4.6	380 399 396	(392) 10.2		23 23 29	(25) 3.5	12 7 5	(8) 3.6
+	50	128 107 129	(121) 12.4	13 8 14	(12) 3.2	345 381 394	(373) 25.4		29 29 28	(29) 0.6	7 16 7	(10) 5.2
+	150	110 137 114	(120) 14.6	8 11 11	(10) 1.7	422 424 425	(424) 1.5		21 29 33	(28) 6.1	6 11 4	(7) 3.6
+	500	116 119 134	(123) 9.6	5 12 13	(10) 4.4	399 405 361	(388) 23.9		31 C 29	(30) 1.4	6 3 5	(5) 1.5
+	1500	76 85 92	(84) 8.0	11 7 9	(9) 2.0	434 380 376	(397) 32.4		36 22 25	(28) 7.4	7 5 6	(6) 1.0
+	5000	46 46 37	(43) 5.2	5 5 7	(6) 1.2	39 74 44	(52) 18.9		15 12 21	(16) 4.6	3 7 1	(4) 3.1
Positive controls S9-Mix +	Name Concentration (µg/plate) No. colonies per plate	2AA		2AA		DAN			BP		2AA
		1		2		10			5		2
		1099 1410 1350	(1286) 165.0	236 292 243	(257) 30.5	1135 1103 1090	(1109) 23.2		383 477 536	(465) 77.2	876 723 678	(759) 103.8

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

#            Standard deviation

C               Contaminated

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Ammonium thioglycolate was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Ammonium thioglycolate was tested in a bacterial gene mutations assay performed following a protocol compliant with the OECD guideline # 471. The direct plate incorporation procedure was performed with Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535 and TA 1537 at concentrations up to 5,000 µg/plate. Cytotoxic effects were observed in the absence and in the presence of a metabolic activator (Aroclor 1254-induced rat liver S9) at the concentration of 5,000 µg/plate. Ammonium thioglycolate did not induce mutations in the bacterial mutation test in either the absence or presence of metabolic activator in any strain tested. The positive and negative controls included in the experiment showed the expected results.