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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative

HPRT: negative

MNT in vitro: negative

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
his operon for S. typhimurium strains
trp operon for the E. coli 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
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.
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

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
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.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Qualifier:
according to guideline
Guideline:
other: EU method B.49 (In vitro Mammalian Cell Micronucleus Test)
Principles of method if other than guideline:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487. The optimum in responses was found with the time schedule stated in the Summary.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: BASF, D035-2017-DMPA
- Expiration date of the lot/batch: 2020-09 26
- Purity test date: Jun 28 - Aug 29, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under storage conditions: guaranteed over the study period

OTHER SPECIFICS
- measurement of pH, osmolality, and precipitate in the culture medium to which the test chemical is added: yes, pH was adjusted, if necessary, in the stock solutions to physiological values using a small amount of 2M HCL.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: primary human lymphocytes (collected from healthy non-smoking donors)
- Normal cell cycle time: app. 16h

For lymphocytes:
- Sex, age and number of blood donors: 1male (20 years), 1 female (29 years)
- Whether whole blood or separated lymphocytes were used: whole blood in medium
- Whether blood from different donors were pooled or not: no
- Mitogen used for lymphocytes: PHA

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
285, 489, 872 µg/ml
Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μl/mL, whichever is the lowest. With regard to the molecular weight of the test item, 872 μg/mL (approx. 10 mM) were applied as top concentration, as no cytotoxicity or precipitation was observed.
Vehicle / solvent:
culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2 (4h, and 20h exposure)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 48h, stimulation with PHA
- Exposure duration/duration of treatment: 4h (with and without S9) or 20h (only without S9)
- Harvest time after the beginning of treatment: 40h (2 - 2.5 cell cycles)
4h treatment, 16h recovery + 20h incubation with Cytochalasin B after removal of test substance
20h treatment + 20h incubation with Cytochalasin B after removal of test substance

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- If cytokinesis blocked method was used for micronucleus assay: indicate the identity of cytokinesis blocking substance (e.g. cytoB), its concentration, and duration and period of cell exposure: Cytochalasin, 4µg/ml, 20h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes. 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended carefully. After removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold. The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa, mounted after drying and covered with a coverslip.
- Number of cells spread and analysed per concentration: at least 1000 binucleated cells per culture
- Criteria for scoring micronucleated cells: According to Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytokinesis-block proliferation index
- Any supplementary information relevant to cytotoxicity: at least 5000 cells per cultrue used for evaluation
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
A test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In this study, no precipitation of the test item in the culture medium was observed at the end of treatment.
No relevant influence on osmolarity was observed. The pH was adjusted to physiological values using a small amount of 2 M HCl
In this study in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.

Table 1     Summary of results
Exp. Preparation Test item Proliferation Cytostasis Micronucleated  
interval concentration index in %* cells 95% Ctrl limit
    in µg/mL CBPI   in %**  
Exposure period 4 h without S9 mix
I 40 h Solvent control1 1.73   0.25 0.01 – 1.20
Positive control2 1.59 19.5 9.55S 2.66 – 22.74
285 1.71 3.2 0.25
498 1.80 n.c. 0.40
    872 1.73 n.c. 0.60  
Trend test: p-value 0.053
Exposure period 20 h without S9 mix
II 40 h Solvent control1 1.86   0.30 0.00 – 1.14
Positive control3 1.45 48.0 4.00S 1.15 – 6.44
93.0 1.72 15.6 0.20
285 1.68 21.2 0.60
    872 1.66 23.1 0.50  
Trend test: p-value 0.414
Exposure period 4 h with S9 mix
I 40 h Solvent control1 1.83   0.75 0.00 – 1.24
Positive control4 1.37 55.1 5.00S 1.01 – 7.34
285 1.76 8.1 0.75
498 1.67 19.6 1.10
    872 1.76 8.7 0.50  

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S The number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 Culture medium 2 MMC 0.8 μg/mL 3 Demecolcine 125 ng/mL 4 CPA 17.5 μg/mL

Conclusions:
Dimethyl(propyl)amine did not induce micronuclei in primary human lymphocytes in vitro.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
July 2016
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch number of test material: D035-2017-DMPA
- Expiration date of the lot/batch: 2020-09-26
- Purity - study number: 17L00248


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Absence of Mycoplasma contamination: yes
- Cell cycle length: 12-16h
- Modal number of chromosomes: 20
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS). All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity up to approximate confluence and subcultured twice weekly.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
47.6, 85.7, 154.3, 277.8, 500, 900µg/mL
Repeat experiment: 300, 450, 600, 750, 900µg/mL
The top dose equals 10mM, the maximum concentration according to current guidelines, since no cytotoxicity, changes in pH value or osmolality, nor precipitation was observed in a pre-test up to 9.2mM (800µg/mL)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium

- Justification for choice of solvent/vehicle: good solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration:2
- Number of independent experiments : 3 (2 with S9, 2 w/out S9)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20x10^6 / 40mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20-24h
- Exposure duration/duration of treatment: 4h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 5-7 days
- Selection time (if incubation with a selective agent): app. 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days
- Selective agent: 6-thioguanine (10µg/mL)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^6 cells / 20mL

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency; relative survival (RS)
Evaluation criteria:
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent negative control.

Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Species / strain:
Chinese hamster Ovary (CHO)
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:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: small amounts of 37% HCl were added to the stock solution to adjust the pH value
- Data on osmolality: not affected by test substance
- Precipitation and time of the determination: none
- Cell morphology: not influenced by treatment

Repeat experiments:
Experiment 1 was aborted due to a severe shift in the pH value
In experiment 2, a significant increase in mutant frequency in a single concentration (not highest dose) compared to concurrent control was observed. This part was repeated though all obtained values were well within historical control values.
Experiment 3 (repeat experiment) was accidentally carried out without S9 mix
Experiment 4 was the correct repeat experiment.

Experiment 1: aborted due to pH shift

Experiment 2

Test groups [µg/mL] S9 # of colonies Mutant frequency (per 10^6)
    flask 1 flask 2 uncorrected corrected
Negative control - 2 6 2.00 2.89
154.3 - 0 4 1.00 1.48
277.8 - 0 2 0.50 0.91
500.0 - 6 4 2.50 2.67
900.0 - 2 3 1.25 2.18
EMS 400.0 - 93 80 43.25 94.54
Negative control + 1 1 0.50 0.75
154.3 + 2 0 0.50 0.91
277.8 + 1 4 1.25 1.91
500.0 + 6 5 2.75 4.20
900.0 + 5 4 2.25 3.60
DMBA 1.25 + 55 58 28.25 55.67

Experiment 3

Test groups [µg/mL] S9 # of colonies Mutant frequency (per 10^6)
    flask 1 flask 2 uncorrected corrected
Negative control - 2 6 2.00 3.42
450.0 - 1 1 0.50 0.65
600.0 - 3 1 1.00 1.77
750.0 - 3 1 1.00 1.62
900.0 - 3 1 1.00 1.70
EMS 400.0 - 92 102 48.50 127.63

Experiment 4

Test groups [µg/mL] S9 # of colonies Mutant frequency (per 10^6)
flask 1 flask 2 uncorrected corrected
Negative control + 1 2 0.75 0.89
450.0 + 0 0 0.00 0.00
600.0 + 3 8 2.75 3.29
750.0 + 2 2 1.00 1.33
900.0 + 3 1 1.00 1.65
DMBA 1.25 + 67 72 34.75 59.91

correction on the basis of the absolute cloning efficiency 2 at the end of the expression period

significant values indicated in bold

Conclusions:
In the absence and the presence of metabolic activation, Dimethyl(propyl)amine is not a mutagenic substance in the HPRT locus assay using CHO cells.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro genotoxicity:

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 (BASF AG 40M0164/994067). 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 the second preincubation test conducted without S9 mix. In none of the tests any mutagenic effect could be detected, indicating that DMPA is not mutagenic in bacteria.

In a study according to OECD 476 (BASF SE 2020), DMPA was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phophoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells. The highest tested concentration was based on the molecular weight of the test substance corresponding to 10mM. THe cells were treated with DMPA for 4 hours in the absence or presence of rat liver S9. No relevant cytotoxicity was observed. DMPA did not increase the mutant frequencies with or without S9. Positive and negative controls were valid. Thus, DMPA is not mutagenic in the HPRT locus assay.

The potential of DMPA to induce micronuclei in human lymphocytes was assessed in two independent experiments in vitro according to OECD 487 (BASF 2020). Cells were either exposed without metabolic activation (rat liver S9) for 4 or 24 hours, or in the presence of S9 mix for 4 hours. For each experimental group, 1000 binucleated cells each from two parallel cultures were analyzed for cytogenetic damage. The highest applied concentration was based on the molecular weight of the test substance and corresponds to app. 10mM. No cytotoxicty was observed up to the highest concentration. No relevant increase in the numbers of micronucleated cells was observed in the presence or absence of metabolic activation. Appropriate positive control substances led to the expected significant increase in micronuclei.

Data for the structurally related substance DMEA (598 -56 -1) are comparable:

Ames: neg.

HPRT: neg.

CA: neg.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No 1272/2008. All mutagenicity / genotoxicity tests conducted with DMPA (Ames, HPRT, MNT) gave negative results. As a result, DMPA does not need to be classified as mutagenic under Regulation (EC) No 1272/2008, as amended for the thirteenth time in Regulation (EU) No 2018/1480.