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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 June 1999 - 14 July 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Dimethyl(propyl)amine
EC Number:
213-139-9
EC Name:
Dimethyl(propyl)amine
Cas Number:
926-63-6
Molecular formula:
C5H13N
IUPAC Name:
dimethyl(propyl)amine
Details on test material:
- Name of test material (as cited in study report): Dimethylpropylamin
- Physical state: liquid, colourless, clear
- Analytical purity: 99.4% (GC)
- Lot/batch No.: K 322/1
- Storage condition of test material: room temperature, under N2



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
Third experiment (preincubation test without metabolic activation): 0, 125, 250, 500, 1000 and 1500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
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:
4-nitroquinoline-N-oxide
Remarks:
5 µg/plate, in DMSO, for E. coli WP2 uvrA Migrated to IUCLID6: (4-NQO)
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.
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:
in preincubation test only (for detail see 'additional information on results')
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation: no precipitation detected
Cytotoxicity: no cytotoxic effects were seen in the standard plate-incorporation test with and without metabolic activation. In the tests with preincubation but without metabolic activation cytotoxicty occurred at
1.) 1500 µg/ plate (ratio of number of revertants for test and control data < 0.5 ) for strain TA98
2.) 5000 µg/ plate (ratio of number of revertants for test and control data < 0.5 ) for strain E. coli WP2
3.) 2500 µg/ plate (reduced background growth) for strains TA 1535, TA 100, TA 1537 and TA 98
In the tests with preincubation and metabolic activation cytotoxicty occurred at 5000 µg/plate (reduced background growth) for strains TA 1535, TA 100, TA 1537 and TA 98

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 18 120 11 32 27
20 17 119 12 28 25
100 19 133 13 28 25
500 17 118 13 25 26
2500 18 120 13 29 28
5000 20 116 17 27 33
Positive control 689 1252 988 1218 1028
SPT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 19 131 17 41 28
20 19 135 17 32 26
100 19 130 12 35 27
500 21 139 11 32 27
2500 15 118 11 36 25
5000 14 126 12 33 30
Positive control 203 1088 141 1070 202
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 20 125 11 28 27
20 17 132 10 27 24
100 18 144 13 25 36
500 17 136 9 24 32
2500 B B B B 24
5000 B B B B 13
Positive control 1174 1082 759 1230 558
PIT with S9-Mix
 [mean no. of mutations/ plate]
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98 WP2 uvrA
Solvent control 19 136 10 40 37
20 17 140 10 37 35
100 18 136 9 31 39
500 16 132 11 32 45
2500 16 132 9 30 44
5000 B 75 3 25 35
Positive control 100 640 96 722 244
Experiment 3: PIT without S9-mix
Dosage [µg/ plate] TA 1535 TA 100 TA 1537 TA 98
Solvent control 20 136 8 27
125 19 124 10 30
250 19 117 7 20
500 15 118 8 19
1000 16 110 7 23
1500 15 92 7 14
Positive control 1027 1177 717 1158
B: reduced background growth, cytotoxicity

Applicant's summary and conclusion

Conclusions:
A test of bacterial gene mutagenicity was conducted for DMPA according to the OECD TG 471 (1997), with following strains: 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, and for the first preincubation test with and without S9 mix. The concentrations were 125, 250, 500, 1000 and 1500 µg/plate for second preincubation test conducted 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.