Carbamic acid, N-[[5-[[[2-[2-(dimethylamino)ethoxy]ethoxy]carbonyl]amino]-1,3,3-trimethylcyclohexyl]methyl]-, 2-[2-(dimethylamino)ethoxy]ethyl ester

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:

15 Apr - 06 Jun 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: "Comission Directive 2000/32/EC, L1362000, Annexe 4D", dated May 19, 2000

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA, "The salmonella typhimurium reverse mutation assay"

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Kanpoan No. 287 - EPA; Eisei No. 127 - Ministry of Health & Welfare; Heisei 09/10/31 Kikyoku No.2 - Ministry of Intern. Trade & Industry

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Landwirtschaft und Forsten, Mainzer Straße 90, D-65189 Wiesbaden, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

441-100-8

EC Name:

-

Cas Number:

351197-46-1

Molecular formula:

Hill formula: C24 H48 N4 O6 CAS formula: C24 H48 O6 N4

IUPAC Name:

2-[2-(dimethylamino)ethoxy]ethyl N-{[1,3,3-trimethyl-5-(9-methyl-2-oxo-3,6-dioxa-1,9-diazadecan-1-yl)cyclohexyl]methyl}carbamate

Constituent 2

Reference substance name:

Carbamic acid, [[5-[[[2-[2- (dimethylamino)ethoxy]ethoxy]carbonyl]amino]-1,3,3- trimethylcyclohexyl]methyl]-,2-[2- (dimethylamino)ethoxy]ethyl ester

IUPAC Name:

Carbamic acid, [[5-[[[2-[2- (dimethylamino)ethoxy]ethoxy]carbonyl]amino]-1,3,3- trimethylcyclohexyl]methyl]-,2-[2- (dimethylamino)ethoxy]ethyl ester

Details on test material:

- Name of test material (as cited in study report): UAX-1179
- CAS No.: 351197-46-1
- Physical state / appearance: pale, amber liquid
- Analytical purity: >99% proprietary tertiary amines
- Lot/batch No.: M. 17-08-01 (declared on the label)
- Expiration date of the lot/batch: September 19, 2003
- Stability in solvent: stable in distilled water
- Storage condition of test material: room temperature, in tightly closed bottle under nitrogen
- Other: On the day of the experiment, the test item UAX-1179 was dissolved in deionised water.
The solvent was chosen because of its solubility properties. The solution was neutralised with 2N HCI.
No precipitation of the test item occurred up to the highest investigated dose.

Method

Target gene:

his-operon, trp-operon
TA 1537 his C 3076; rfa-; uvrB-
TA 98 his D 3052; rfa-; uvrB-, R-factor
TA 1535 his G 46; rfa-; uvrB-
TA 100 his G46; rfa-; uvrB-, R-factor
WP2 uvrA trp-; uvrA-

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-Naphthoflavone induced rat liver S9 from 8-12 weeks old male Wistar Han: IBM rats.

Test concentrations with justification for top dose:

Concentrations in experiment I: Plate Incorporation Test (with and without S9 mix): 33, 100, 333, 1000, 2500, 5000 µg/plate
Concentrations in experiment II: Pre-Incubation Test (with and without S9 mix): 33, 100, 333, 1000, 2500, 5000 µg/plate
Concentration range in the pre test was 3 - 5000 µg/plate

Vehicle / solvent:

Solvent: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100 without S9

Migrated to IUCLID6: 10 µg/plate

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 10 µg/plate 4-nitro-o-phenylene-diamine in TA 98, and 50 µg/plate in TA1537

Remarks:

TA1537 and TA98 without S9

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

WP2 uvrA without S9

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 2.5 µg/plate

Remarks:

TA 1535, TA1537, TA98 and TA100 with S9

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene 10 µg/plate

Remarks:

WP2 uvrA with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: plate incorporation; Experiment II: preincubation

EXPERIMENTAL PERFORMANCE
The following materials were mixed in a test tube and poured onto the minimal agar plates:
100 µl Test solution at each dose level, solvent (negative control) 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 (cf. test system, pre-culture of the strains),
2000µl Overlay agar

The test item UAX-1179 was dissolved in deionised water. The solution was neutralised with 2N HCI.

DURATION
- Preincubation period: 60 minutes
- Selection time (if incubation with a selection agent): 48 hours

SELECTION AGENT (mutation assays): minimal agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth / regular background growth / spontaneous reversion rates in the negative and solvent control

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding 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.

Statistics:

No statistical evaluation of the data is required.

Results and discussion

Test results

Test results 1

Species / strain:

bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100 and Escherichia coli strain WP2 uvrA

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

Positive controls validity:

valid

Test results 2

Species / strain:

bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98, and TA 100 and Escherichia coli strain WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 at 5000 µg/plate without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation, except, for strain TA 1537 where a minor toxic effect was observed at 5000 µg/plate without S9 mix in Experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, either in the presence or absence of metabolic activation. There was also no tendancy of higher mutation rates with increasing concentrations in the range below the genarally acknowledged border of biological relevence.

In Experiment II, the data in the negative control of strain TA 98 was slightly above the historical control range (with S9 mix). Since this deviation is rather small (44 colonies versus 43 colonies), this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

The historical range of positive controls was exceeded without metabolic activation in strains TA 1535 (Exp. I) and TA 100 (Exps. I and II). This effect indicates the sensitivity of the strains rather than compromising the assay.

Remarks on result:

other: other: experiment I: plate incorporation test

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of experiment I (Test dates from April 22, 2002)

With or without S9 mix	Test substance concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution type						Frame shift type
		TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
S9 mix (-)	Solvent control	161 164 153	159	17 11 10	13	62 60 46	56	29 31 32	31	5 6 7	6
	33	202 156 158	172	9 12 6	9	48 57 60	55	37 27 28	31	13 8 10	10
	100	168 188 176	177	7 7 9	8	58 62 61	60	35 33 27	32	8 4 7	6
	333	196 173 177	182	12 8 9	10	55 68 62	62	23 29 39	30	9 10 9	9
	1000	192 204 191	196	10 10 6	9	58 52 57	56	33 35 39	36	8 6 5	6
	2500	202 152 173	176	11 7 7	8	48 54 58	53	31 26 27	28	8 8 10	9
	5000	172 141 174	162	8 12 9	10	54 55 48	52	35 17 35	29	10 11 5	9
S9 mix (+)	Solvent control	178 212 204	198	11 9 10	10	49 50 46	48	43 39 44	42	12 15 12	13
	33	163 154 206	174	10 8 14	11	66 58 56	60	44 46 54	48	8 12 11	10
	100	213 145 208	189	14 11 13	13	65 54 52	57	42 43 51	45	13 15 13	14
	333	187 186 191	188	12 11 15	13	62 60 66	63	49 49 44	47	10 9 13	11
	1000	197 218 174	196	9 7 10	9	59 67 56	61	52 57 55	55	12 11 18	14
	2500	203 200 215	206	13 11 14	13	55 65 59	60	38 34 48	40	7 11 13	10
	5000	191 180 189	187	10 12 8	10	58 65 53	59	43 43 45	44	10 7 12	10
Positive control not requiring S9 mix	Name	sodium azide		sodium azide		MMS		4-NOPD		4-NOPD
	Concentration (mg/plate)	10		10		5 µL/plate		10		50
	Number of colonies/plate	1310 1288 1114	1237	878 782 880	847	840 824 863	842	237 243 226	235	49 66 55	57
Positive control requiring S9 mix	Name	2-AA
	Concentration (mg/plate)	2.5		2.5		10		2.5		2.5
	Number of colonies/plate	1048 1142 1175	1122	192 224 238	218	270 305 284	286	608 639 554	600	95 87 80	87

Table 2: Results of experiment II (Test dates from June 03, 2002)

With or without S9 mix	Test substance concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution type						Frame shift type
		TA 100		TA 1535		WP2 uvrA		TA 98		TA 1537
S9 mix (-)	Solvent control	134 150 152	145	9 12 16	12	60 51 56	56	26 30 26	27	10 8 9	9
	33	144 153 136	144	11 11 7	10	60 55 55	57	25 20 27	24	13 10 9	11
	100	152 148 125	142	11 15 15	14	52 48 52	51	34 37 27	33	9 10 9	9
	333	129 141 136	135	14 9 16	13	59 42 43	48	30 32 25	29	9 10 6	8
	1000	146 154 173	158	11 11 14	12	59 50 47	52	23 18 26	22	8 10 5	8
	2500	149 124 132	135	12 10 16	13	51 58 56	55	30 26 25	27	8 12 13	11
	5000	147 135 145	142	8 6 6	7	53 52 54	53	29 21 20	23	5 3 4	4
S9 mix (+)	Solvent control	176 202 217	198	14 9 10	11	65 62 63	63	30 32 29	30	16 17 15	16
	33	167 204 192	188	7 11 9	9	58 67 57	61	41 33 40	38	15 9 14	13
	100	180 186 203	190	9 16 9	11	66 63 60	63	36 24 31	30	10 15 9	11
	333	168 159 180	169	8 14 14	12	55 48 52	52	29 27 33	30	11 16 15	14
	1000	138 158 130	142	9 11 6	9	59 56 49	55	30 35 32	32	12 16 14	14
	2500	145 150 128	141	10 11 13	11	53 49 52	51	35 35 32	34	10 13 8	10
	5000	166 171 178	172	9 12 10	10	57 59 65	60	38 37 30	35	11 14 13	13
Positive control not requiring S9 mix	Name	sodium azide		sodium azide		MMS		4-NOPD		4-NOPD
	Concentration (mg/plate)	10		10		5 µL/plate		10		50
	Number of colonies/plate	807 870 911	863	814 770 829	804	444 507 457	469	241 230 219	230	51 56 52	53
Positive control requiring S9 mix	Name	2-AA
	Concentration (mg/plate)	2.5		2.5		10		2.5		2.5
	Number of colonies/plate	526 597 541	555	137 164 165	155	301 299 359	320	294 298 288	293	52 49 55	52

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 bacteria strains used.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

There's no evidence for genotoxic potential of UAX-1179.

Executive summary:

UAX-1179 was tested in an Ames test using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and the Escherichia coli strain WP2 uvrA . The substance was tested up to 5000 µg/plate with and without metabolic activation. No toxic effects occurred in the test groups, except for strain TA 1537, where a minor toxic effect was observed at 5000 µg/plate without S9 mix. Under the experimental conditions the test item did not induce mutations by base pair changes or frameshifts in the genome of the strains used.