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Toxicological information

Genetic toxicity: in vitro

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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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Butyldiethanolamin S
- Physical state: liquid, colourless to yellowish
- Analytical purity: 99.7% (Gas Chromatography)
- Lot/batch No.: 13-8583
- Stability: 2 years and 7 months after date of manufacture (analytical characterisation performed in Nov 1996); stability was guaranteed during study period
- 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
Controlsopen allclose all
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
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
Positive controls:
yes
Remarks:
without S9-mix
Positive control substance:
9-aminoacridine
Remarks:
100 µg/plate, in DMSO, for TA 1537 Migrated to IUCLID6: (AAC)
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
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 2mL 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:
Migrated from field 'Test system'. 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 (migrated information): 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.