2,2'-butyliminodiethanol

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:

04 March 1997 - 24 April 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP with slight deviation from current requirements: as positive control substance in the presence of S9 mix, only 2-aminoanthracene (2-AA) was tested.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-butyliminodiethanol

EC Number:

203-055-0

EC Name:

2,2'-butyliminodiethanol

Cas Number:

102-79-4

Molecular formula:

C8H19NO2

IUPAC Name:

2-[butyl(2-hydroxyethyl)amino]ethan-1-ol

Details on test material:

- Name of test material (as cited in study report): Butyldiethanolamin S
- Physical state: liquid, colourless to yellowish
- Storage condition of test material: room temperature

Method

Target gene:

his operon for S. typhimurium strains
trp operon for the E. coli strain

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: TA 98: rfa-, uvrB-, R-factor; TA 100: rfa-, uvrB-, R-factor; TA 1535: rfa-, uvrB-; TA 1537: rfa-, uvrB; WP2: trp-; uvr A-

Metabolic activation:

with and without

Metabolic activation system:

S9 liver mix prepared from Sprague-Dawley rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First experiment (standard plate test, with and without metabolic activation): 0, 20, 100, 500, 2500 and 5000 µg/plate
Second experiment (preincubation test with and without metabolic activation): 0, 20, 100, 500, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: good solubility of the test item in the selected vehicle

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(sterility control)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9-mix

Positive control substance:

other: 2-aminoanthracene (2-AA)

Remarks:

2.5 µg/plate, in DMSO, for TA 1535, TA 1537, TA 100, TA 98; 60 µg/plate, in DMSO, for E. coli WP2 uvrA

Controls 2

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)

Remarks:

5 µg/plate, in DMSO, for TA 1535 and TA 100

Controls 3

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

9-aminoacridine

Remarks:

100 µg/plate, in DMSO, for TA 1537 (AAC)

Controls 4

Positive controls:

yes

Remarks:

without S9-mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

10 µg/plate, in DMSO, for E. coli WP2 uvrA

Controls 5

Positive controls:

yes

Remarks:

(without S9-mix)

Positive control substance:

other: 4-nitro-o-phenylendiamine (NOPD)

Remarks:

10 µg/plate, in DMSO, for TA 98

Details on test system and experimental conditions:

In the standard plate test, tubes were filled with 2 mL portions of soft agar and kept in a water bath at 45 °C. This soft agar consisted of 100 mL agar and 10 mL amino acid solution. As amino acid solution for the soft agar was used 0.5 mM histidine and 0.5 mM biotin for TA strains and 0.5 mM tryptophan for the E. coli strain.
Then following components are added:
0.1 mL test solution or vehicle
0.1 mL fresh bacterial culture
0.5 mL S9 -mix or phosphate buffer
After mixing samples were poured onto Vogel-Bonner (minimal glucose agar plates) plate and incubated for 48 - 72 hrs in the dark at 37 °C.

For the preincubation test 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL of the S9 mix were incubated at 37 °C for 20 minutes. After addition of 2 mL soft agar samples were poured onto agar plates and incubated again at 37 °C for 48 to 72 hrs.
For the E. coli strain, plate test differed again in mixture of amino acid solution of the soft agar, the histidine component used for the TA strains being replaced by tryptophan.
Triplicate testing is done.
After incubation, the his+ revertant colonies of bacteria were counted.

Evaluation criteria:

An assay is accepted when the following criteria are met:
1.) number of colonies in the negative control is in the historical control range
2.) no indication of bacterial contamination (checked by sterility control)
3.) number of colonies in the positive controls are in the range of historical control data
4.) titer of viable bacteria is ≥ 10 E+9/mL

Toxicity is detected by:
1.) decrease in the number of revertants
2.) titer reduction
3.) clearing or diminution of the background lawn

Precipitation:
As long as no interference between precipitation and colony counting occurs is 5 mg/plate set as maximum dose even for relatively insoluble compounds.

A test chemical is to be considered as mutagenic when:
1.) increase of number of revertant colonies is reproducible and dose-related.
2.) in at least 1 tester strain doubling of colony counts with or without S-9 mix or after adding a metabolizing system is seen.

A test chemical is to be considered as non-mutagenic when:
1.) the number of revertants is inside the range of historical negative control data in 2 experiments performed independently from each other.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

no increase in number of revertants was observed

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decrease in revertants number reported for S. typhimurium TA 1535 at about 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation: no precipitation detected

Remarks on result:

other: all strains/cell types tested

Remarks:

Remarks: tested in the standard plate and the preincubation test

Any other information on results incl. tables

Experiment 1: Standard plate-incorporation test

SPT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	21	118	14	26	35
20	18	120	11	31	35
100	19	127	9	27	32
500	16	113	10	27	35
2500	19	100	9	30	30
5000	18	114	10	25	30
Positive control	1109	1111	397	1227	652
SPT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	22	119	13	42	43
20	17	109	14	53	36
100	18	125	12	45	39
500	19	107	11	44	40
2500	15	79	14	48	38
5000	14	90	11	42	33
Positive control	141	1066	118	1035	233
Experiment 2:
Preincubation test PIT without S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	20	158	13	33	33
20	18	162	11	32	32
100	18	154	11	33	32
500	16	129	10	32	31
2500	14	130	9	30	29
5000	12	132	10	30	28
Positive control	1278	1580	672	1079	791
PIT with S9-Mix  [mean no. of mutations/ plate]
Dosage [µg/ plate]	TA 1535	TA 100	TA 1537	TA 98	WP2 uvrA
Solvent control	20	158	11	47	42
20	19	166	12	46	39
100	17	154	13	44	33
500	15	140	11	43	34
2500	13	127	10	44	29
5000	11	134	8	40	28
Positive control	126	1081	139	1002	217

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
CLP: not classified
DSD: not classified

Executive summary:

Butyldiethanolamine was tested in bacterial gene mutation test using Salmonella typhimurium starins TA 98, TA 100, TA 1535 and TA 1537 and E. coli WP2 uvrA . The test concentrations were 20, 100, 500, 2500 and 5000 µg/plate for the standard plate test with and without S9 mix, as well as for the preincubation test with and without S9 mix. In none of the tests any mutagenic effect could be detected. Therefore the substance is to be considered as non-mutagenic in bacteria.