Carbonic acid, compound with 2-aminoethanol (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental phase of this study was performed between 13 May 2008 and 04 November 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of GLP inspection: 19-08-2008 Date of Signature on GLP certificate:04-03-2009

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Carbonic acid, compound with 2-aminoethanol (1:2)

EC Number:

244-600-2

EC Name:

Carbonic acid, compound with 2-aminoethanol (1:2)

Cas Number:

21829-52-7

Molecular formula:

C2H7NO.1/2CH2O3

IUPAC Name:

carbonic acid - 2-aminoethanol (1:2)

Constituent 2

Reference substance name:

MEA-carbamate

IUPAC Name:

MEA-carbamate

Test material form:

liquid: viscous

Details on test material:

-Sponsor's identification :MEA-carbamate
-Description : Pale amber coloured extremely viscous liquid
-Batch number :1314-206
-Date received : 14 April 2008
-Storage conditions :Room temperature in the dark
- Molecular formula (if other than submission substance): HOCH2CH2NH3+-O2C-NHCH2CH2OH
- Molecular weight (if other than submission substance): 166.17
- Analytical purity: 100%
- Purity test date: 12-05-2008
- Expiration date of the lot/batch: 21-11-2008
- Stability under test conditions: Stable under normal temperatures and pressure

Method

Target gene:

Histidine for Salmonella.
Tryptophan for E.Coli

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):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/beta­naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
main test:
Experiment one: 50, 150, 500, 1500 and 5000 µg/plate
Experiment two: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix Migrated to IUCLID6: Benzo(a)pyrene: 5 µg/plate

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 mix Migrated to IUCLID6: 4-Nitroquinoline-1-oxide: 0.2 µg/plate

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water.

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 3 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 5 µg/plate

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

Sterile distilled water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix Migrated to IUCLID6: N-ethyl-N'-nitro-N-nitrosoguanidine: 2 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): Not applicable

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

Evaluation criteria:

Acceptance 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.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 109 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should not be an excessive loss of plates due to contamination.

Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Standard deviation

Results and discussion

Test results

Test results 1

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

Tested up to maximum recommended dose of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

COMPARISON WITH HISTORICAL CONTROL DATA:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory).

Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

RESULTS

Preliminary ToxicityTest

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA^-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The numbers of revertant colonies for the toxicity assay were:

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	120	86	90	107	98	136	111	107	118	102	100
+	TA100	93	103	99	118	101	108	106	103	114	96	99
-	WP2uvrA^-	32	21	31	23	21	30	31	21	24	29	26
+	WP2uvrA^-	37	40	36	31	25	26	21	21	18	22	24

MutationTest

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). These data are not given in the report. The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile.

Results for the negative controls (spontaneous mutation rates) are presented inTable1and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5 with the results also expressed graphically in Figure 1 to Figure 4.

Information regarding the equipment and methods used in these experiments as required by the Japanese Ministry of Economy, Trade and Industry and Japanese Ministry of Health, Labour and Welfare are presented in Appendix 1.

A history profile of vehicle and positive control values is presented in Appendix 4.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1               Spontaneous Mutation Rates (Concurrent Negative Controls

Range-finding Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
128		25		40		19		15
129	(132)	25	(23)	35	(41)	19	(20)	14	(15)
140		19		47		22		15

Main Test

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA^-		TA98		TA1537
147		24		35		11		20
147	(149)	27	(28)	35	(31)	21	(18)	12	(14)†
154		34		24		22		11

 

Table2               Test Results: Range-Finding Test– Without Metabolic Activation

Test Period		From: 24 May 2008					To: 27 May 2008
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		WP2uvrA‑			TA98		TA1537
-	0	137 100 106	(114) 19.9#	24 22 20	(22) 2.0	34 30 30		(31) 2.3	16 18 19	(18) 1.5	12 10 12	(11) 1.2
-	50	111 114 109	(111) 2.5	23 18 18	(20) 2.9	43 30 27		(33) 8.5	22 13 20	(18) 4.7	21 13 11	(15) 5.3
-	150	121 114 114	(116) 4.0	23 29 23	(25) 3.5	25 24 37		(29) 7.2	18 19 14	(17) 2.6	11 10 13	(11) 1.5
-	500	123 109 111	(114) 7.6	26 29 25	(27) 2.1	36 29 35		(33) 3.8	19 19 20	(19) 0.6	14 14 10	(13) 2.3
-	1500	132 112 139	(128) 14.0	23 34 23	(27) 6.4	36 27 27		(30) 5.2	16 15 16	(16) 0.6	16 10 10	(12) 3.5
-	5000	96 109 110	(105) 7.8	25 19 29	(24) 5.0	32 34 31		(32) 1.5	20 22 20	(21) 1.2	7 13 11	(10) 3.1
Positive controls   S9-Mix   -	Name Concentration (µg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		1513 1127 1049	(1230) 248.5	114 119 120	(118) 3.2	651 660 647		(653) 6.7	115 115 114	(115) 0.6	2572 2606 2770	(2649) 105.9

 

 

Table3               Test Results: Range-Finding Test– With Metabolic Activation

Test Period		From: 24 May 2008					To: 27 May 2008
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		WP2uvrA‑			TA98		TA1537
+	0	106 120 78	(101) 21.4#	16 11 12	(13) 2.6	36 36 30		(34) 3.5	35 22 40	(32) 9.3	29 24 13	(22) 8.2
+	50	102 82 103	(96) 11.8	8 9 18	(12) 5.5	49 33 35		(39) 8.7	26 42 29	(32) 8.5	23 21 19	(21) 2.0
+	150	114 101 101	(105) 7.5	14 13 20	(16) 3.8	41 41 33		(38) 4.6	22 29 25	(25) 3.5	16 18 19	(18) 1.5
+	500	115 115 120	(117) 2.9	18 15 20	(18) 2.5	56 38 38		(44) 10.4	30 30 35	(32) 2.9	19 13 12	(15) 3.8
+	1500	125 106 101	(111) 12.7	13 18 21	(17) 4.0	35 44 45		(41) 5.5	36 42 34	(37) 4.2	14 23 22	(20) 4.9
+	5000	86 91 96	(91) 5.0	15 16 23	(18) 4.4	43 45 44		(44) 1.0	29 36 38	(34) 4.7	25 16 12	(18) 6.7
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2370 2221 2043	(2211) 163.7	319 354 307	(327) 24.4	443 514 510		(489) 39.9	126 151 114	(130) 18.9	207 158 206	(190) 28.0

 

 

Table4               Test Results: Main Test– Without Metabolic Activation

Test Period		From: 31 May 2008 07 June 2008†					To: 03 June 2008 10 June 2008†
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		WP2uvrA‑			TA98		TA1537†
-	0	126 132 146	(135) 10.3#	25 23 24	(24) 1.0	30 30 34		(31) 2.3	16 16 13	(15) 1.7	18 21 21	(20) 1.7
-	50	110 126 128	(121) 9.9	19 27 25	(24) 4.2	30 34 36		(33) 3.1	18 14 19	(17) 2.6	18 24 16	(19) 4.2
-	150	119 95 139	(118) 22.0	25 18 33	(25) 7.5	38 23 33		(31) 7.6	16 18 22	(19) 3.1	26 20 18	(21) 4.2
-	500	128 119 122	(123) 4.6	31 26 30	(29) 2.6	29 34 34		(32) 2.9	16 19 16	(17) 1.7	27 14 23	(21) 6.7
-	1500	141 120 150	(137) 15.4	20 19 31	(23) 6.7	40 34 25		(33) 7.5	22 15 13	(17) 4.7	16 16 20	(17) 2.3
-	5000	137 128 125	(130) 6.2	20 21 22	(21) 1.0	41 20 45		(35) 13.4	14 14 16	(15) 1.2	20 24 25	(23) 2.6
Positive controls   S9-Mix   -	Name Concentration (μg/plate) No. colonies per plate	ENNG		ENNG		ENNG			4NQO		9AA
		3		5		2			0.2		80
		345 344 344	(344) 0.6	84 180 189	(151) 58.2	488 497 482		(489) 7.5	124 144 140	(136) 10.6	377 362 1651	(797) 739.9

 

 

Table5               Test Results: Main Test– With Metabolic Activation

Test Period		From: 31 May 2008 07 June 2008†					To: 03 June 2008 10 June 2008†
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		WP2uvrA‑			TA98		TA1537†
+	0	129 146 147	(141) 10.1#	11 12 12	(12) 0.6	44 30 33		(36) 7.4	26 20 23	(23) 3.0	18 18 19	(18) 0.6
+	50	129 133 120	(127) 6.7	8 7 7	(7) 0.6	35 32 32		(33) 1.7	24 26 21	(24) 2.5	12 13 13	(13) 0.6
+	150	115 110 133	(119) 12.1	10 9 9	(9) 0.6	21 31 29		(27) 5.3	23 26 22	(24) 2.1	20 23 16	(20) 3.5
+	500	123 123 132	(126) 5.2	8 10 14	(11) 3.1	46 22 36		(35) 12.1	22 20 29	(24) 4.7	18 15 21	(18) 3.0
+	1500	126 90 118	(111) 18.9	9 10 13	(11) 2.1	31 30 31		(31) 0.6	14 32 19	(22) 9.3	19 14 18	(17) 2.6
+	5000	135 109 139	(128) 16.3	3 8 9	(7) 3.2	42 41 32		(38) 5.5	23 19 20	(21) 2.1	27 18 12	(19) 7.5
Positive controls   S9-Mix   +	Name Concentration (μg/plate) No. colonies per plate	2AA		2AA		2AA			BP		2AA
		1		2		10			5		2
		2915 2403 3244	(2854) 423.8	213 255 255	(241) 24.2	564 556 578		(566) 11.1	163 172 167	(167) 4.5	307 392 298	(332) 51.9

 

†            Experimental procedure repeated at a later date (presence and absence of S9) due to high solvent counts in original test

#            Standard deviation

PLEASE SEE ATTACHED 1) Figures1 -4 Dose-Response Curves. 2) Pages 23 -30 (Appendix 1 Report of Results in Mutagenicity Test using Micro-organisms)

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA^-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

Results.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

The test material was considered to be non-mutagenic under the conditions of this test.