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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames: negative for S. typhimurium (TA1535, TA100, TA1537, TA98) (BASF 1993, Zeiger 1982) and for E. coli (WP2 uvrA) (BASF 2022)


CA: negative (Loveday 1990)
Mouse lymphoma: ambiguous (Barber 2000)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Remarks:
preliminary results, the RSS wil be updated when a final report is available
Adequacy of study:
key study
Study period:
August 2021
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:
26 Jun 2020
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
The S9 fraction was prepared according to Ames et al. at BASF SE in an AAALACapproved laboratory in accordance with the German Animal Welfare Act and the effective European Council Directive (experimental conduct with records and documentation in general accordance with the GLP principles, but without GLP status).
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany GmbH) received 80 mg/kg b.w. phenobarbital i.p. and beta-naphthoflavone orally (both supplied by Sigma-Aldrich, 82024 Taufkirchen, Germany) each on three consecutive days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The day/night rhythm was 12 hours: light from 6 am to 6 pm and darkness from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum. 24 hours after the last administration, the rats were sacrificed, and the induced livers were prepared using sterile solvents and glassware at a temperature of +4°C. The livers were washed with 150 mM KCl solution. Afterwards, the livers were weighed and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, appropriate portions of the supernatant (S9 fraction) were stored at -70°C
to -80°C. The preparation date and protein content of the S9 fraction were given in the Appendix (S9 Fraction characterization).

The S9 mix was prepared freshly prior to each experiment. For this purpose, a sufficient amount of S9 fraction was thawed at room temperature and 1 part of S9 fraction is mixed with 9 parts of S9 supplement (cofactors). This mixture of both components (S9 mix) was kept on ice until used. The concentrations of the cofactors in the S9 mix were:
MgCl2 8 mM
KCl 33 mM
glucose-6-phosphate 5 mM
NADP 4 mM
phosphate buffer (pH 7.4) 15 mM
The phosphate buffer is prepared by mixing a Na2HPO4 solution with a NaH2PO4 solution in a ratio of about 4:1.
To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.
Test concentrations with justification for top dose:
In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose at least in the 1st Experiment. However, this maximum dose was tested even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate or > 5 μL/plate might also be tested in repeat experiments for further clarification/substantiation.

1st Experiment
Strains: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Standard plate test with and without S9 mix
Number of plates: 3 test plates per dose or per control

2nd Experiment
Strains: E. coli WP2 uvrA
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Type of test: Preincubation test with and without S9 mix
Number of plates: 3 test plates per dose or per control
Reason: No mutagenicity was observed in the standard plate test.
Vehicle / solvent:
Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Negative solvent / vehicle controls:
yes
Remarks:
The vehicle control with and without S9 mix only contains the vehicle used for the test substance at the same concentration and volume for all tester strains. Vehicle controls were used for several BASF projects done in parallel. For details see raw data.
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
other: 2-aminoanthracene (2-AA)
Remarks:
Sterility control
Additional plates were treated with soft agar, S9 mix, buffer, vehicle and the test substance but without the addition of tester strain.
Details on test system and experimental conditions:
TEST SUBSTANCE PREPARATIONS
The test substance was weighed and topped up with the chosen vehicle to achieve the required concentration of the stock solution.
The test substance was dissolved in DMSO. To achieve a clear solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
The further concentrations were diluted according to the planned doses.
All test substance formulations were prepared immediately before use.

ANALYSIS OF TEST SUBSTANCE PREPARATION
The stability of the test substance in the vehicle DMSO was not determined analytically, because the test substance was administered immediately after preparation and is usually stable.

TEST SYSTEM
For testing, deep-frozen (-70°C to -80°C) bacterial culture (E. coli WP2 uvrA) was thawed at room temperature, and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in the shaking water bath at 37°C for about 12 - 16 hours. The optical density of the fresh bacteria culture was determined.
Fresh culture of bacteria was grown up to late exponential or early stationary phase of growth (approximately 10^9 cells per mL). This culture grown overnight was kept in iced water from the beginning of the experiment until the end in order to prevent further growth.
The use of the strain mentioned was in accordance with the current scientific recommendations for the conduct of this assay.
The Escherichia coli strain was obtained from Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA on 02 Dec 2014.

Checking the tester strain
E. coli WP2 uvrA was checked for UV sensitivity.
Histidine and tryptophan auxotrophy were checked in each experiment via the spontaneous rate.

Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) was based on the method of Ames et al..

Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation)
or
0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

Preincubation Test
The experimental procedure was based on the method described by Yahagi et al. and Matsushima et al..
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker.
Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.
Evaluation criteria:
Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat
experiments for further clarification/substantiation.
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No relevant increase in the number of trp+ revertants and no bacteriotoxic effect was observed in the standard plate test and in the preincubation assay under all test conditions.
Test substance precipitation was observed with and without S9 mix at 5000 μg/plates.
The additional treated plates for sterility control showed no contamination in all performed experiments.
Conclusions:
Under the experimental conditions chosen here, it is concluded that Dimethylphthalate is not a potent mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of external metabolic activation.
Executive summary:

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).
The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the respective historical control data of each tester strain. In addition, the positive control substances induced a significant increase in the number of revertant colonies with and without S9 mix. This increase was within the range of the respective historical control 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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 471 under GLP conditions and is reliable with restrictions. These restrictions are: only 4 Strains of Salmonella typhimurium were tested, strain TA102 is missing.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Palatinol M (DMP)
- Physical state: colorless liquid
- Stability under test conditions: analytically verified
- Storage condition of test material: RT
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 mix
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO for test substance and controls
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: + S9 mix: -2 .5 ug 2-aminoanthracene (2-AA) for t TA 100, TA 98, TA 1537 and TA 1535 - S9 mix: 5 μg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) for the strains TA 100 and TA 1535; 10 μg 4-nitro-o-phenylendiamine (NPD) TA98; 100 μg 9-aminoacridine chloride
Details on test system and experimental conditions:
METHOD OF APPLICATION:
standard plate test and preincubation method

DURATION
- Preincubation period: 20min at 37°C
- Exposure duration: 48h after incubation at 37°C in the dark bacterial colonies are counted

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 2 experiments, 3 test plates per dose and per control
Evaluation criteria:
Criteria for positive evaluation in the Ames test:
- doubling of the spontaneous rate mutation rate (control)
- dose-response relationsship
- reproducibility of the results
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation found

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- weak bacteriotoxic effect - reduced his- background growth, slight decreas in the number of his+ revertants at 5000 µg/plate dose

Table 1: Results of the Ames test

(mean number of revertants +-SD)

 

Dose level in µg/plate

TA1535

TA100

TA1537

TA98

Experiment 1 without metabolic activation

Solvent

19+-5

124+-11

7+-1

24+-2

20

19+-3

132+-9

8+-2

28+-5

100

18+-4

128+-7

7+-3

23+-0

500

16+-3

132+-22

6+-1

18+-3

2500

19+-6

135+-12

6+-3

20+-2

5000

17+-4

119+-8

7+-2

18+-1

Positive control

868+-76

748+-78

788+-27

926+-116

-

Experiment 2 without metabolic activation

Solvent

18+-3

115+-8

7+-1

22+-3

20

19+-2

99+-14

7+-4

21+-3

100

15+-2

106+-7

7+-3

23+-2

500

16+-2

108+-9

8+-2

20+-3

2500

15+-3

82+-17

5+-1

20+-2

5000

7+-2

64+-10

4+-1

15+-2

Positive control

782+-62

745+-81

700+-10

1065+-97

-

Experiment 1 with metabolic activation

Solvent

19+-2

137+-4

10+-3

39+-3

20

15+-2

124+-13

9+-1

46+-2

100

16+-1

137+-14

7+-2

29+-5

500

18+-2

131+-5

9+-2

23+-6

2500

16+-2

115+-15

9+-1

23+-4

5000

14+-2

145+-4

9+-2

23+-2

Positive control

106+-9

1093+-25

102+-5

637+-13

-

Experiment 2 with metabolic activation

Solvent

18+-3

128+-6

12+-3

40+-2

20

12+-2

120+-7

10+-2

36+-4

100

13+-3

115+-14

8+-1

36+-1

500

11+-2

93+-2

9+-2

36+-2

2500

10+-2

100+-14

7+-1

27+-2

5000

7+-1

36+-10

7+-1

17+-4

Positive control

106+-14

659+-32

103+-9

678+-86

 

SUMMARY
The substance Palatinol M (DMP) was tested for mutagenicity
in the Ames test (standard plate test and preincubation
test) both in the presence and in the absence of a metabolizing system obtained from rat liver (S-9 mix) using the strains TA 1535, TA 100, TA 1537 and TA 98.

In the standard plate test as well as in the preincubation
test no increase in the number of his+ revertants was observed when tested in the tester strains TA 1535, TA 100, TA 1537 and TA 98, both in the presence and absence of S9.

Toxicity:
A weakly bacteriotoxic effect (reduced his- background
growth, slight decrease in the number of his+ revertants)
was observed in the preincubation test at 5000 ug/plate.

According to the results of the present study, the test
substance Palatinol M (DMP) is not mutagenic in the Ames
test under the experimental conditions chosen here.
Conclusions:
The test substance was not mutagenic in the Ames test.
Executive summary:

The study is reliable with restrictions and was conducted according to OECD TG 471 (Bacterial reverse mutation assay) under GLP conditions. Genetic toxicity in vitro was tested in 4 strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98) instead of five (minor restriction: TA102 is missing). The study was conducted before OECD 471 was adopted in 1997 (strain TA102 was then included into the OECD TG). No enhanced number of revertants was observed in presence or absence of S9 rat liver mix.

The test substance was not mutagenic in the Ames test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is reliable with restrictions and is fully suffucient for endpoint evaluation. The study was conducted equivalent or similar to OECD TG 473 (In vitro mammalian chromosome aberration test). GLP status is not clear and minor deviations are restrictions. Basic data given, acceptable publication which meets basic scientific principles
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
not specified
Principles of method if other than guideline:
Method: other: Chromosome aberration test in mammalian cells in vitro
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dimethyl phthalate
- Analytical purity: 99%
- Purity test date: analysis provided by supplier (Aldrich)
- Lot/batch No.: not reported
- Stability under test conditions: not reported
- Storage condition of test material: nor reported
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy´s 5A (modified) medium (KC Biological, Lenexa, KS), buffered with 20mM HEPES (Aldrich Chemical Co., Milwaukee, WI) and supplemented with 10% FCS (KC, Biological, Lenexa, KS) 2mM L-glutamine, 50IU/ml peniciline, and 50µg/ml streptomycin (Gibco, Grand Island, NY)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes/no
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
10.5 hr incubation: 150, 498 and 1500 ug/ml;
12.0 hr incubation: 498, 1500 and 4980 ug/ml;
20.5 hr incubation: 3060, 4080 and 5100 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: no preincubation
- Exposure duration: 12h/10h acitvated/non-activated chromosomal aberration test, and longer incubation (see doses and table1 for details)
- Expression time (cells in growth medium): 24h incubation after culture initiation, 2h treatment with test substance + S9 mix and 8h treatment with fresh medium/8h treatment with testsubstance -S9 mix, 2h colcemid treatment, cytotoxicity analsis and harvest/fixation/staining of cells
- Fixation time (start of exposure up to fixation or harvest of cells): 4h after start of treatment, 2h after colcemid stain

SPINDLE INHIBITOR (cytogenetic assays): colcemid (24h treatment)
STAIN (for cytogenetic assays):
- HOECHST 33258 (0.5µg/ml in phosphate buffer,pH 7.3); fixation in 3:1 methanol:glacial acetic acid (2h after treatment with colcemid); analysis in flourescence microcopes to analyse the frequency of metaphase cells after cycling 1x/2x times in BrdU (frequency of first division M1, and second division M2), cell cycle delay was analysed
- stain for evaluation of chromosomal aberration: 5% GIEMSA staining

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: 200 cells per dose

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: estimation of confluence of cell monolayer in treated flask in comparison with control falsks and noting the presence of mitotic cells.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: cell cycle delay
Evaluation criteria:
Chromosomal aberration
- simple: defined as chromatid gap, break, fragment, and deletion or chomosome gap, break or double minutes
- complex: defined as interstitial deletions, triradials, quadriradials, rings and dicentric chromosomes
- other: defined as pulverized chromosome or cells with greater than 10 aberrations
REMARK: chromatide and chromosome gaps were recorded but not included in the totals of the statistical analysis

Evalution of AB as percent of total cells
Statistics:
- binominal sampling assumption described by Margolin et al. (1983)
- Dunnett´s method for multiple dose comparisonsStatistical tests were performed on both the dose response curve and
individual dose points. A statistically significant (P < 0.05) difference for one dose point and a significant trend (P < 0.015) were considered
weak evidence of a positive response; significant differences for two or more doses indicate the trial is positive.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test substance was not mutagenic in the CHO in vitro chromosome aberration test.
Executive summary:

The study is reliable with restrictions and was conducted as an NTP test equivalent or similar to OECD TG 473 (In vitro mammalian chromosome aberration test), GLP status is not clear. In vitro cytogenicity was tested in Chinese hamster ovary cells in vitro. No enhanced chromosomal aberrations were observed in presence or absence of S9 rat liver mix at different harvest times of cells and doses (10.5h incubation: 150, 498 and 1500µg/ml;

12.0h incubation: 498, 1500 and 4980µg/ml; 20.5h incubation: 3060, 4080 and 5100µg/ml). Under conditions of this study, dimethyl phthalate was not mutagenic in the chromosome aberration assay at doses up to 5100 mg/ml.

The test substance was not mutagenic in the CHO in vitro chromosome aberration test.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study is sufficient for endpoint evaluation. GLP-study, well documented and scientifically acceptable. Performed before guideline 476 was established.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Mouse Lymphome Forward Mutation Assay was perforemd. Assay determines the ability of the test substance to induce forward mutations at the Thymidine Kinase (TK) locus as assayed by colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT). Method is based on Clive et al. (1979).
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dimethyl Phthalate
- Physical state: clear colorless liquid
- Storage condition of test material: at room temperature in the dark
Target gene:
thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 supplemted with F68, L-glutamine, sodium pyruvate, antibiotics and horse serum (10% w/v); cloning medium = medium additionally supplemented with agar 0.35% to chieve a semisolid state; selection medium = cloning medium additionally supplemented with 3µg/ml TFT
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes ( selection against TK-/- phenotypeic cells with methotrexat 8 days prior to use)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and necessary cofactors; S9 consists of the 9000x g supernatant prepared from Arochlor 1254-induced adult male rat livers, batches are checked for sterility, CYP1A1 activity and proteine content.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 140 µl/ml in acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: with metabolic activation: 4h
- Exposure duration: 2 days
- Selection time (if incubation with a selection agent): 10-14 days: analysis of colonies

SELECTION AGENT (mutation assays): TFT, 5-trifluorothymidine

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:
- day 1 after treatment: 24h after treatment celles are adjusted to a density of 3x10E5/ml to maintain optimal growth
- day 2 after treatment: determination of cell counts and celection of cultures for cloning and mutant selection

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth over a 2day period (compared to solvent control)

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:
Evaluation criteria:
- mutant frequency is calculated by dividing the total number of colonies in each set of three mutant selection dishes.
- cells are exposed to a range of concentrations of test materials that span the cellular processes from no observed toxicity to complete lethality within 24h of treatment.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effect - range fomr7.21 - 7.28
- Effects of osmolality: no details
- Water solubility: stock solution acetone, prior to experiments, dilutions in medium were prepared, test material was incompletely soluble and formed oily droplets at concentration above 100nl/ml medium (without metabolic activation) and 500nl/ml medium (with metabolic activation)

RANGE-FINDING/SCREENING STUDIES:
yes, initial range finding studies for dose selection (coverage of a range of toxicity fomr no observed toxicity to complete lethality)


Table 1: results from the in vitro mutangenicity test in mouse lymphoma cells

Treatment

Suspension growth

Total mutant colonies

Total viable colonies

Cloning efficiency

Relative growth (%)

Mutant frequency

(10E-6 Units)

Nonactivation

Solvent control

11.2

48.0

441.0

76.8

100.0

20.8

Solvent control

11.9

51.0

538.0

89.7

100.0

18.9

Solvent control

10.1

66.0

443.0

73.8

100.0

29.8

Solvent control

9.7

47.0

453.0

75.5

100.0

20.7

EMS 0.25 µl/ml

5.0

765.0

412.0

68.7

40.6

371.3

EMS 0.40 µl/ml

6.3

877.0

347.0

57.8

42.5

505.5

Growth of TC relative to solvent control (%)

TC 400 nl/ml

93.6

108.0

497.0

104.8

98.1

43.5

TC 400 nl/ml

82.3

74.0

522.0

110.1

90.6

28.3

TC 500 nl/ml

41.8

87.0

369.0

77.8

32.5

47.1

TC 500 nl/ml

43.3

90.0

368.0

77.6

33.7

48.9

TC 650 nl/ml

48.1

85.0

358.0

75.5

36.3

47.5

TC 650 nl/ml

51.8

81.0

353.0

74.5

38.6

45.9

TC 700 nl/ml

45.3

73.0

357.0

75.3

34.1

40.9

TC 700 nl/ml

43.2

58.0

295.0

62.2

26.9

39.3

S9-Activation

Solvent control

15.9

126.0

626.0

104.3

100.0

40.2

Solvent control

13.4

103.0

670.0

111.7

100.0

30.7

Solvent control

16.7

115.0

648.0

108.0

100.0

35.5

Solvent control

15.6

109.0

590.0

98.3

100.0

36.9

MCA 2.5 µl/ml

8.8

564.0

385.0

64.2

34.7

293.0

MCA 4.0 µl/ml

7.2

575.0

397.0

66.2

29.4

289.7

Growth of TC relative to solvent control (%)

TC 100 nl/ml

72.5

250.0

697.0

110.0

79.8

71.7

TC 100 nl/ml

64.6

233.0

706.0

111.4

72.0

66.0

TC 200 nl/ml

74.3

271.0

685.0

108.1

80.3

79.1

TC 200 nl/ml

58.9

247.0

650.0

102.6

60.4

76.0

TC 400 nl/ml

60.6

330.0

599.0

94.5

57.3

110.2

TC 400 nl/ml

64.8

244.0

681.0

107.5

69.7

71.6

TC 500 nl/ml

53.2

269.0

629.0

99.3

52.8

85.5

TC 500 nl/ml

48.1

294.0

579.0

91.4

44.0

101.5

TC 600 nl/ml

31.4

590.0

310.0

48.9

15.4

380.6

TC 600 nl/ml

29.2

513.0

305.0

48.1

14.0

336.4

EMS   Ethylmethan sulfonate

TC       Test compound

MCA   3-methylcholanthrene

Executive summary:

Gene mutation in vitro was tested by Hazleton Biotechnology Company (1986) in a Mouse Lymphome Forward Mutation Assay equivalent or similar to OECD TG 476 (In vitro mammalian cell gene mutation assay). This assay determines the ability of the test substance to induce forward-mutations in the Thymidine Kinase (TK) locus as assayed by colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT). A problem in this study was that test material was incompletely soluble and formed oily droplets at concentration above 100nl/ml medium (without metabolic activation) and 500nl/ml medium (with metabolic activation). Under nonactivation conditions (200 nl/ml 500 nl/ml), non detectable to moderate toxicities were detected (percentage relative growth: 101.7% to 52.5%). The mutant frequency exceeding 52.3 x 10 -6 was required. The assayed treatments induced mutant frequencies ranging from 21.2 x 10 -6 to 48.4 x 10 -6 . There was no evidence for mutagenic activity of the test material, but highly toxic treatments were not achieved. Another nonactivation assay was therefore performed (400 nl/ml to 700 nl/ml). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 43.8 x 10 -6 . Treatments at 500 nl/ml and 650 nl/ml induced small but significant increases in the mutant frequency. However, higher toxic treatments at 700 nl/ml were inactive. The test material was evaluated as nonmutagenic without activation in this assay. In the presence of metabolic activation (100 nl/ml to 600 nl/ml) were analyzed for mutant induction; low to high toxicities were induced (percent relative growths, 80.3% to 14.0% ). The minimum criterion for mutagenesis in this assay was a mutant frequency exceeding 63.7 x 10- 6; all of the assayed treatments induced significant increases in the mutant frequency. The increases ranged from 1.8-fold to 10.6 -fold above the background mutant frequency (average of solvent controls). The test material was therefore considered mutagenic with activation in this assay.

Test substance was mutagenic with metabolic activation at high doses (600 nl/ml medium) in the in vitro mouse lymphoma mutation assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MNT i.p.: negative (NTP 1986, 1994)
SCE: negative (Yuschenko 1977)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to standrad NTP protocols for mammalian testing of chromosome aberration (micronucleus formation) and is comparable to OECD TG 475. The study is reliable with acceptable restrictions: GLP-status is unclear, doses are applied three times every 24h, limited documentation (online file).
Principles of method if other than guideline:
Method: other: NTP standard protocol (Micronucleus in mouse bone marrow)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dimethyl phthalate
Species:
mouse
Strain:
B6C3F1
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
route of exposure: intraperitoneal injection
Duration of treatment / exposure:
72 h; 3 x i.p.
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
Dose / conc.:
2 250 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
No. of animals per sex per dose:
5M per treatment and 4M in vehicle/positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 25mg/kg bw
Tissues and cell types examined:
bone marrow and erythrocytes are examined for presence of micronuclei
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- Treatment: 3 treatments every 24h, duration 72h
- Sampling: 24h after last dosing
- The bone marrow is flushed from the femurs and spread onto slides.

DETAILS OF SLIDE PREPARATION:
- The slides are air-dried, fixed and stained with a fluorescent DNA-specific dye that illuminates any micronuclei that may be present.


Statistics:
Formal statistical analysis:
- trend test (cells containing micronuclei)
- pairwise comparison of each dose group to the corresponding control
- Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): yes, positive control was valid
- Ratio of PCE/NCE (for Micronucleus assay): no
- Appropriateness of dose levels and route: yes
- Statistical evaluation: yes

Table1:

Start Date

Sample Collection Time

Sex

Cell

Dosing Regimen

Trend Test P-Value

03/15/1994

24 Hours

Male

PCE

IP x 3, 72 Hours

0.166

 

Dose (mg/kg)

No. of Animals Scored

Mean MN-PCE/1000 PCE ± SEM

Pairwise P

Vehicle Control:

Corn Oil

0         

4

1.00 ± 0.00

 

Test Chemical:

 

750         

5

0.90 ± 0.33

0.586

 

1500         

5

1.20 ± 0.30

0.344

 

2250         

5

0.70 ± 0.30

0.756

 

3000         

5

1.60 ± 0.29

0.137

Positive Control:

Cyclophosphamide

25         

4

3.63 ± 0.69

0.000

Conclusions:
Interpretation of results (migrated information): negative
No chromosome aberration was detected in male B6C3F1 mice at doses up to 3000 mg/kg bw.
Test substance was not mutagenic.
Executive summary:

The study was conducted according to standard NTP protocol for mammalian bone marrow chromosome aberration testing of chemicals which is comparable to OECD Guideline 475. GLP

status is unclear. Acceptable restriction: doses are applied three times every 24h, limited documentation (online file).

Male B6C3F1 mice were exposed to dose levels of 0, 750, 1500, 2250 and 3000 mg/kg bw (5 animals/treatment) three times every 24h (72h duration) via intraperitoneal injection. 24h after the last application bone marrow was analyzed for presence of micronuclei in polychromatic erythrocytes (PCE). No mutagenic effect of the test substance was reported. The relevant negative (vehicle: corn oil) and positive (cyclophosphamide) controls were valid.

No chromosome aberration was detected in male B6C3F1 mice at doses up to 3000 mg/kg bw.

Test substance was not mutagenic.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was conducted according to standard NTP protocols for mammalian testing of chromosome aberration (micronucleus formation) and is comparable to OECD TG 475. The study is reliable with acceptable restrictions: GLP-status is unclear, doses are applied three times every 24h, limited documentation (online file).
Principles of method if other than guideline:
Method: other: NTP standard protocol (Micronucleus in rat bone marrow)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Dimethyl phthalate
Species:
rat
Strain:
Fischer 344
Sex:
male
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Details on exposure:
route of exposure: intraperitoneal injection
Duration of treatment / exposure:
72 h; 3 x i.p.
Dose / conc.:
3 000 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
Dose / conc.:
4 000 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
Dose / conc.:
5 000 mg/kg bw/day (nominal)
Remarks:
intraperitoneal injection
No. of animals per sex per dose:
5M per treatment
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneal injection
- Doses / concentrations: 25mg/kg bw
Tissues and cell types examined:
bone marrow and erytrocytes are examined for presence of micronuclei
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- Treatment: 3 treatments every 24h, duration 72h
- Sampling: 24h after last dosing
- The bone marrow is flushed from the femurs and spread onto slides.

DETAILS OF SLIDE PREPARATION:
- The slides are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present.


Statistics:
Formal statistical analysis:
- trend test (cells containing micronuclei)
- pairwise comparison of each dose group to the corresponding control
- Data are typically presented as the mean number of micronucleated cells per 1,000 cells for each treatment group
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Start Date

Sample Collection Time

Sex

Cell

Dosing Regimen

Trend Test P-Value

12/14/1993

24 Hours

Male

PCE

IP x 3, 72 Hours

0.046

 

Dose (mg/kg)

No. of Animals Scored

Mean MN-PCE/1000 PCE ± SEM

Pairwise P

Vehicle Control:

Corn Oil

0         

5

0.80 ± 0.25

 

Test Chemical:

 

3000         

5

1.00 ± 0.52

0.390

 

4000         

4

2.88 ± 1.63

0.024

 

5000         

3

2.00 ± 0.29

0.108

Positive Control:

Cyclophosphamide

25         

5

11.60 ± 4.90

0.0001

Conclusions:
No chromosome aberration was detected in male F344 rats at doses up to 5000 mg/kg bw.
Test substance was not mutagenic.
Executive summary:

The study was conducted according to standard NTP protocol for mammalian bone marrow chromosome aberration testing of chemicals which is comparable to OECD Guideline 475. GLP

status is unclear. Acceptable restriction: doses are applied three times every 24h, limited documentation (online file).

Male F344 rats were exposed to dose levels of 0, 3000, 4000 and 5000 mg/kg bw (5 animals/treatment) three times every 24h (72h duration) via intraperitoneal injection. 24h after the last application bone marrow was analyzed for presence of micronuclei in polychromatic erythrocytes (PCE). No mutagenic effect of the test substance was reported. The relevant negative (vehicle: corn oil) and positive (cyclophosphamide) controls were valid.

No chromosome aberration was detected in male F344 rats at doses up to 5000 mg/kg bw.

Test substance was not mutenic.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
other: Only secondary literature. Original publication in Russian language.
Principles of method if other than guideline:
Method: other: no data
GLP compliance:
no
Type of assay:
sister chromatid exchange assay
Species:
mouse
Strain:
not specified
Sex:
not specified
Route of administration:
intraperitoneal
Duration of treatment / exposure:
single dose
Dose / conc.:
1 400 other: mg/kg
Remarks:
single dose, intraperitoneal
Sex:
not specified
Genotoxicity:
negative
A single intraperitoneal administration of dimethyl phthalate to mice in 
the dose of 1400 mg/kg (1/5 of LD50) did not entail any pronounced 
cytogenetic effect on the cells of the bone marrow. 
No significant increase in the incidences of chromosome
aberrations or SCE in the 900 bone marrow metaphases
examined.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro studies: Bacterial systems


Genetic toxicity in vitro was tested in 4 strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98) according to OECD (Bacterial reverse mutation assay) under GLP conditions  [BASF AG, 1993]. No enhanced number of revertants was observed in presence or absence of S9 rat liver mix (Arochlor 1254 induced). Concentrations tested range were 20, 100, 500, 2500, 5000 µg/plate. Thus, the test substance was not mutagenic in bacteria. Both positive control and negative control were valid.


These findings are supported by data from Zeiger et al. [1982] where no mutagenic activity was observed at doses up to 6666 µg/plate in the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic (S9 mix, Arochlor 1254 induced) activation.


Further, preliminary results from genetic toxicity testing in E. coli WP2 uvrA according to OECD (Bacterial reverse mutation assay) under GLP conditions are available [BASF SE, 2022]. No enhanced number of revertants was observed in presence or absence of S9 rat liver mix. The tested concentration range was 33 - 5000 µg/plate. Both positive control and negative control were valid. Thus, under the experimental conditions of the study, the test substance was not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of external metabolic activation.


 


Agarval (1985) also failed to detect any relevant increase in the number of revertant colonies up to 2000 µg/plate in Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538, and TA2637. A concurrently performed spot test was also negative in all strains. DMP proved toxic to TA100, where background growth was reduced to 12% at the highest concentration. No details for the other strains were reported.


 


Two further abstract were found in the literature:
Seed (1982) detected a weakly positive response in TA100 (no other strains tested) in a suspension assay. This interpretation is not shared by the registrant due to several limitations in the study and a very weak response at severe cytotoxicity. Furthermore, the above described well documented studies do not show a concern for a mutagenic potential in this strain.


 


No increase in the number of revertants in TA 98 were observed in a pre-incubation assay by Sato (1994).


 


In vitro studies: Mammalian cell systems


Gene mutation in vitro was tested by Hazleton Biotechnology Company [1986] in a Mouse Lymphome Forward Mutation Assay equivalent or similar to OECD TG 476 (In vitro mammalian cell gene mutation assay). This assay determines the ability of the test substance to induce forward-mutations in the Thymidine Kinase (TK) locus as assayed by colony growth of L5178Y TK+/- mouse lymphoma cells in the presence of 5-trifluorothymidine (TFT). The problem of this study was that test material was incompletely soluble and formed oily droplets at concentration above 100 nl/ml medium (without metabolic activation) and 500 nl/ml medium (with metabolic activation).


Under non-activation conditions toxicity determined as relative growth ranged from 98.1% at 400nl/ml to 26.9% at 700nl/ml. Treatments at 500 nl/ml and 650 nl/ml induced small but significant increases in the mutant frequency. However, higher toxic treatments at 700 nl/ml were inactive. The test material was evaluated as non-mutagenic without activation in this assay.


In the presence of metabolic activation relative growth ranged from app 80% to 14.0% at the highest concentration of 600nl/ml. A relevant increase of mutant colonies app. 10.6 -fold above the average background mutant frequency was only observed for the highest concentration of 600nl/ml. Because this increase was only observed when the relative growth was reduced to 14-15%, it is not considered clear evidence of mutagenicity. The assay is evaluated as equivocal.


 


In vitro cytogenicity was tested by Loveday et al. [1990] in Chinese hamster ovary cells equivalent or similar to OECD TG 473 (In vitro mammalian chromosome aberration test). No enhanced numbers of chromosomal aberrations compared to controls were observed in presence or absence of S9 rat liver mix at different harvest times of cells and doses (10.5 h incubation: 150, 498 and 1500 µg/ml; 12.0 h incubation: 498, 1500 and 4980 µg/ml; 20.5 h incubation: 3060, 4080 and 5100 µg/ml). Under the conditions of this study, test substance was not mutagenic in the chromosome aberration assay at doses up to 5100 mg/ml.


 


This result was confirmed by Tsuchiya in 1976, who did not detect an increase in chromosomal aberrations in human leukocytes after treatment with 0.02mg/ml DMP.


 


Loveday (1990) also evaluated SCEs in CHO cells. With metabolic activation there was a weak increase compared to concurrent control values (30%), but which was in the range of control values reported for assays with other chemicals. Due to a dose-dependent trend the assay was evaluated as equivocal. No increase was observed without metabolic activation.


 


Jaerventaus et al. [1984] also evaluated SCEs in CHO cells, but did not detect an increase in the number of SCEs with or without metabolic activation.


 


DMP was also tested in a cell transformation assay using Balb/c 3T3 cells (TSCAT 1985). None of the frequencies of transformed foci observed for the test material treatments were significantly altered and no evidence of a dose-related response was observed. The positive control was valid.


 


In vivo studies


Male F344 rats were exposed to dose levels of 0, 3000, 4000 and 5000 mg/kg bw (5 animals/treatment) three times every 24 h (72 h duration) via intraperitoneal injection [NTP, 1986]. The study was conducted according to standard NTP protocol for mammalian bone marrow chromosome aberration testing of chemicals which is comparable to OECD Guideline 475. 24 h after the last application bone marrow was analyzed for presence of micronuclei in polychromatic erythrocytes (PCE). No mutagenic effect of the test substance was reported. The relevant negative (vehicle: corn oil) and positive (cyclophosphamide) controls were valid.


 


In a second test by NTP [1986], male B6C3F1 mice were exposed to dose levels of 0, 750, 1500, 2250 and 3000 mg/kg bw (5 animals/treatment) three times every 24 h (72 h duration) via intraperitoneal injection. No increase in micronuclei was detected.


 


Additionally, several Rodent Dominant Lethal Assays (Yurchenko 1977, dermal, 1250mg/kg; Yurchenko 1980, i.p., 1250mg/kg; Sheftel 1981, dermal, up to 2000mg/kg) indicate that DMP is not mutagenic in vivo. In the study of Sheftel, a cytogenetic evaluation of the bone marrow was also performed. DMP also had no effect on the number of chromosomal aberrations. This is confirmed by Yurchenko (1977) after single or repeated (1 month) dermal applications of 1250mg/kg. Yurchenko (1977) also did not detect an increase in SCEs in vivo after a single i.p. injection of 1400mg/kg.


 


In a chronic (1 year) dermal carcinogenicity assay, no increase in neoplastic lesions was observed in CD1 -mice at a dose of 2700 mg/kg (NTP 1993, see section on repeated dose for details).

Justification for classification or non-classification

DMP was not mutagenic in bacteria and did not cause chromosomal aberrations in mammalian cells. The questionable increase in SCEs, which was not confirmed in a second in vitro study, is not considered relevant due to several negative assays for chromosomal damage in vitro. Several in vivo studies also confirmed absence of a clastogenic potential. A mouse lymphoma assay yielded ambiguous results only in the presence of metabolic activation, at highly cytotoxic concentrations, which were not soluble anymore and formed oily droplets. There is also no structural alert for DMP. As a conclusion, there is no concern for genotoxicity for DMP.

Classification according to GHS, EU is not required.