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Registration Dossier
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EC number: 231-472-8 | CAS number: 7575-23-7
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
PETMP was negative, with and without metabolic activation, in a complete battery of in-vitro tests.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 September 2008 - 04 January 2009
- 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)
- Version / remarks:
- adopted 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
- Version / remarks:
- August 1998
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- Thymidine kinase
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction from male Wistar rats induced with phenobarbital (80 mg/kg bw) and ß-naphtoflavone (100 mg/kg bw) for three consecutive days by oral route.
- Test concentrations with justification for top dose:
- Experiment I
with metabolic activation: 40, 200, 300, 400, 450, 500, 550, 600 µg/mL
without metabolic activation: 10, 20, 30, 40, 50, 55, 60, 65 µg/mL
Experiment II
with metabolic activation: 130, 180, 230, 280, 480, 530, 630, 675 µg/mL
without metabolic activation: 20, 40, 50, 60, 80, 100, 110, 120 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate 200 and 500 µg/mL, methylmethanesulfonate 10 µg/mL (without metabolic activation)
- Positive control substance:
- other: benzo(a)pyrene 3.5 µg/mL (with metabolic activation)
- Remarks:
- with S9
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4, 24 h
- Expression time (cells in growth medium): 72 or 48 h (5 h or 24 h exposure time)
- Selection time (if incubation with a selection agent): 6 days
SELECTION AGENT (mutation assays): trifluorothymidine (5 µg/mL)
NUMBER OF REPLICATIONS: 4
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
OTHER EXAMINATIONS:
- Other: colony sizing
OTHER: - Evaluation criteria:
- There are several criteria for determining a positive result:
- clear and dose-reIated increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher
than the historical range of negative controls) for at least one of the dose groups.
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low
Large/small colonies ratio (smaller than or equal to 1.5 times the ratio of clastogenic controls MMS and/or B[a]P) is an indication for potential
clastogenic effects rtnd/or chromosomal aberrations. - Statistics:
- not necessary
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at >= 40 µg/mL (-S9); >=313 µg/mL (+S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Interpretation of results: negative with and without activation
PETMP is negative in the mouse lymphoma assay, with and without metabolic activation - Executive summary:
The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cellline L5178Y in accordance with OECD Guideline 476. The se1ection of the concentrations was based on data from the pre-experiment. In experiment I, 600 µg/mL (with metabolic activation) and 65 µg/mL (without metabolic activation) were selected as the highest
concentrations. In experiment II, 675 µg/ml. (with metabolic activation) and 120 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.
The test item was investigated at the following concentrations:
Experiment I
with metabolic activation:
40, 200,300,400, 450, 500, 550, 600 µg/mL
and without metabolic activation:
10, 20, 30, 40, 50, 55, 60, 65 µg/mL
Experiment II
with metabolic activation:
130,180,230,280,480,530,630,675 µg/mL
and without metabolic activation:
20,40,50,60,80,100,110, 120 µg/mL
Growth inhibition was observed in experiment land II with and without metabolic activation.
In experiment I with metabolic activation the relative total growth (RTG) was 13.21% for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 65 µg/mL with a RTG of 16.34%. In experiment II with metabolic activation the relative total growth (RTG) was 13.89% for the highest concentration (675 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 120 µg/mL with a RTG of 10.59%.
In experiment land II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.
Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
The positive controls and showed distinct and biologically relevant effects in mutation frequency.
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2008-09-16 to 2008-02-05
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- May 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- August 1998
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 liver microsomal fraction from male Wistar rats induced with phenobarbital (80 mg/kg bw) and ß-naphtoflavone (100 mg/kg bw) for three consecutive days by oral route.
- Test concentrations with justification for top dose:
- Experiment I:
with metabolic activation: 62.5, 125 and 190 µg/mL
without metabolic activation: 1.95, 3.9 and 7.8 µg/mL
Experiment II:
with metabolic activation: 200, 275 and 300 µg/mL
without metabolic activation: 0.49, 0.98 and 1.95 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with low water solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: ethylmethanesulphonate 400 and 600 µg/mL (without metabolic activation)
- Positive control substance:
- other: cyclophosphamide 0.83 µg/mL (with metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: n.a.
- Exposure duration: 4, 20 h
- Expression time (cells in growth medium): n.a.
- Selection time (if incubation with a selection agent): n.a.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid(R)
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200 metaphases per concentration
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell density
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- There are several criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the concentrations, which is higher than the laboratory negative control range (0.0% - 4.5%
aberrant cells (with metabolic activation) and 0.0% - 4.0% aberrant cells (without metabolic activation)). - Statistics:
- not applicable
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 7.8/190 µg/mL, -/+ S9
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at concentrations of 250 µg/mL and higher.
RANGE-FINDING/SCREENING STUDIES:
According to the used guidelines the highest recommended concentration is 5000 µg/mL. The test item was dissolved in DMSO and diluted in cell
culture medium. The highest concentration evaluated in the preexperiment was 5000 µg/mL. The relative mitotic index was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment-
COMPARISON WITH HISTORICAL CONTROL DATA:
compliant - Conclusions:
- Interpretation of results: negative with and without activation
PETMP does not induce chromosomal aberrations in V79 Chinese hamster lung fibroblasts. - Executive summary:
To investigate the potential of PETMP to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitro chromosome aberration assay was carried out. The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and without metabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and
20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.
The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:
Experiment I:
with metabolic activation: 62.5, 125 and 190 µg/ml,
without metabolic activation: 1.95,3.9 and 7.8 µg/ml,
Experiment II:
with metabolic activation: 200, 275 and 300 µg/ml,
without metabolic activation: 0.49, 0.98 and 1.95 µg/ml.
No precipitation of the test item was noted with and without metabolic activation at all concentrations evaluated in experiment l and II.
In experiment I without metabolic activation, toxic effects of the test item were noted at a concentration of 7.8 µg/ml., with metabolic activation at a concentration of 190 µg/ml;
In experiment II without metabolic activation, toxic effects of the test item were observed at a concentration of 1.95 µg/ml., With metabolic activation, toxic effects of the test item were noted at concentrations of 275 µg/ml, and higher.
In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control.
In experiment I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.
EMS (400 and 600 ug/ml.) and CPA (0.83 ug/ml.) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
In conclusion, it can be stated that during the describedin vitrochromosomal aberration test and under the experimental conditions reported, the test item PETMP did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 03 - 26 July 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- adopted 2000
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9, induced with phenobarbitone/ß-naphthoflavone (80/100 mg per kg per day)
- Test concentrations with justification for top dose:
- 50 to 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: established vehicle for substances with poor water solubility - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- mitomycin C
- other: (without metabolic activation)
- Positive control substance:
- benzo(a)pyrene
- other: 1,8-Dihydroxyanthraquinone, 2-aminoanthracene (with metabolic activation)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Preincubation period: n.a.
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: 3 per concentration per experiment - Evaluation criteria:
- The test material may be considered positive in this test system if the following criteria are met:
The test material should have induced a reproducible, dose-related and statistically (Dunnett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria. - Statistics:
- Dunnett's method of linear regression
- 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
- Species / strain:
- S. typhimurium TA 102
- 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: at 5000 µg/plate
RANGE-FINDING/SCREENING STUDIES:
The test material was non-toxic to the strain of Salmonella used (TA100).
COMPARISON WITH HISTORICAL CONTROL DATA:
pos. and neg. controls within historical range - Conclusions:
- Interpretation of results: negative
PETMP is negative in the Ames test, with and without metabolic activation. - Executive summary:
The genotoxic potential of the test item PETMP was assessed in a Bacterial reverse mutation assay according to OECD Guideline 471 and EU method B13/14.Salmonella typhimuriumstrains TA1535, TA1537, TA102, TA98 and TA100 were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.
The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A white, cloudy precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.
Referenceopen allclose all
Table 1: Experiment I - 4 h exposure - With Metabolic Activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 cells |
Mutation factor |
Quotient large / small colonies |
0 (DMSO) |
100 |
100 |
60.71 |
1 |
1.25 |
40 |
99.45 |
98.54 |
44.95 |
0.74 |
– |
200 |
99.45 |
71.69 |
43.25 |
0.71 |
– |
300 |
96.70 |
62.66 |
68.92 |
1.14 |
– |
400 |
91.76 |
54.59 |
107.48 |
1.77 |
– |
450 |
98.90 |
48.87 |
64.57 |
1.06 |
– |
500 |
89.01 |
32.29 |
112.17 |
1.85 |
0.87 |
550 |
95.60 |
27.59 |
85.69 |
1.41 |
1.26 |
600 |
90.11 |
13.21 |
108.15 |
1.78 |
1.10 |
B[a]P, 3.5 |
98.90 |
68.85 |
243.73 |
4.01 |
0.80 |
B[a]P Benzo[a]pyrene
Table 2: Experiment I - 4 h exposure - Without Metabolic Activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 cells |
Mutation factor |
Quotient large / small colonies |
0 (DMSO) |
100 |
100 |
46.33 |
1 |
2.23 |
10 |
81.87 |
75.76 |
89.19 |
1.93 |
– |
20 |
97.25 |
82.59 |
54.28 |
1.17 |
– |
30 |
95.05 |
69.19 |
43.11 |
0.93 |
– |
40 |
65.60 |
57.56 |
68.01 |
1.47 |
– |
50 |
86.81 |
38.86 |
78.51 |
1.69 |
– |
55 |
100.00 |
34.93 |
57.96 |
1.25 |
1.31 |
60 |
95.60 |
25.04 |
48.16 |
1.04 |
2.19 |
65 |
96.70 |
16.34 |
79.48 |
1.72 |
1.47 |
EMS, 500 |
83.52 |
61.99 |
769.08 |
16.60 |
– |
MMS, 10 |
93.41 |
68.16 |
332.63 |
7.18 |
0.84 |
EMS Ethyl methane sulphonate
MMS Methyl methane sulphonate
Table 3: Experiment II - 24 h Exposure - With Metabolic Activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 cells |
Mutation factor |
Quotient large / small colonies |
0 (DMSO) |
100 |
100 |
95.25 |
1 |
5.45 |
130 |
111.60 |
93.47 |
65.40 |
0.69 |
– |
180 |
111.60 |
86.53 |
46.30 |
0.49 |
– |
230 |
107.84 |
77.71 |
71.18 |
0.75 |
– |
280 |
96.55 |
66.27 |
93.14 |
0.98 |
– |
480 |
114.11 |
40.44 |
49.36 |
0.52 |
– |
530 |
110.97 |
30.48 |
67.17 |
0.71 |
1.39 |
630 |
105.96 |
18.22 |
79.49 |
0.83 |
2.04 |
675 |
89.03 |
13.89 |
164.89 |
1.73 |
2.58 |
B[a]P, 3.5 |
96.55 |
67.52 |
562.23 |
5.90 |
1.06 |
B[a]P Benzo[a]pyrene
Table 4: Experiment II - 24 h exposure - Without Metabolic Activation
Concentration |
Cloning efficiency [%] |
Relative Total Growth [%] |
Mutants per 1E+06 cells |
Mutation factor |
Quotient large / small colonies |
0 (DMSO) |
100 |
100 |
58.93 |
1 |
3.13 |
20 |
92.39 |
95.48 |
109.56 |
1.86 |
– |
40 |
92.39 |
89.64 |
102.58 |
1.74 |
– |
50 |
99.15 |
77.81 |
72.05 |
1.22 |
– |
60 |
95.77 |
57.17 |
79.05 |
1.34 |
– |
80 |
93.52 |
34.35 |
108.22 |
1.84 |
– |
100 |
98.03 |
29.14 |
72.35 |
1.23 |
3.93 |
110 |
103.10 |
22.42 |
82.57 |
1.40 |
3.00 |
120 |
96.90 |
10.59 |
63.38 |
1.08 |
2.15 |
EMS, 200 |
72.68 |
24.95 |
1539.16 |
26.12 |
– |
MMS, 10 |
73.80 |
31.79 |
1207.82 |
20.50 |
0.90 |
EMS Ethyl methane sulphonate
MMS Methyl methane sulphonate
Table 1: Experiment I - 4 h treatment, 20 h fixation - Without Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells |
|
incl. gaps |
excl. gaps |
|||
0 (DMSO) |
100 |
2 |
6 |
3 |
1.95 |
89 |
4 |
8 |
5 |
3.9 |
89 |
6 |
10 |
4 |
7.8 |
20 |
0 |
9 |
4 |
EMS, 600 |
114 |
2 |
27 |
17 |
EMS: Ethyl methane sulphonate
Table 2: Experiment I - 4 h treatment, 20 h fixation - With Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells |
|
incl. gaps |
excl. gaps |
|||
0 (DMSO) |
100 |
0 |
6 |
4 |
62.5 |
94 |
2 |
9 |
5 |
125 |
95 |
1 |
7 |
2 |
190 |
30 |
2 |
14 |
5 |
CPA, 0.83 |
79 |
2 |
35 |
24 |
CPA: Cyclophosphamide
Table 3: Experiment II - 20 h treatment, 20 h fixation - Without Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells |
|
incl. gaps |
excl. gaps |
|||
0 (DMSO) |
100 |
0 |
13 |
7 |
0.49 |
105 |
2 |
8 |
3 |
0.98 |
84 |
2 |
10 |
3 |
1.95 |
32 |
3 |
10 |
4 |
EMS, 400 |
102 |
2 |
24 |
20 |
EMS: Ethyl methane sulphonate
Table 4: Experiment II - 4 h treatment, 20 h fixation - With Metabolic Activation
Concentration |
Mitotic index [%] |
Polyploid cells |
Aberrant cells |
|
incl. gaps |
excl. gaps |
|||
0 (DMSO) |
100 |
2 |
3 |
1 |
200 |
73 |
6 |
14 |
7 |
275 |
39 |
5 |
12 |
4 |
300 |
33 |
5 |
6 |
1 |
CPA, 0.83 |
90 |
2 |
23 |
18 |
CPA: Cyclophosphamide
Table 1: Experiment 1 - Without Metabolic Activation
S9 Mix |
Test substance concentration (µg/ plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
– |
0 |
82 |
25 |
383 |
18 |
12 |
– |
50 |
77 |
22 |
352 |
15 |
10 |
– |
150 |
75 |
22 |
359 |
17 |
7 |
– |
500 |
80 |
21 |
365 |
16 |
9 |
– |
1500 |
71 |
21 |
346 |
18 |
9 |
– |
5000 |
74 P |
33 P |
325 P |
18 P |
7 P |
Pos controls |
Name |
ENNG |
ENNG |
MMC |
4NQO |
9AA |
Conc. (µg/plate) |
3 |
5 |
0.5 |
0.2 |
80 |
|
No. of revertants per plate |
270 |
444 |
1186 |
171 |
1287 |
|
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
MMC Mitomycin C
P Precipitate
Table 2: Experiment 1 - With Metabolic Activation
S9 Mix |
Test substance concentration (µg/ plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
+ |
0 |
87 |
13 |
375 |
46 |
19 |
+ |
50 |
84 |
9 |
365 |
37 |
13 |
+ |
150 |
80 |
10 |
356 |
35 |
18 |
+ |
500 |
77 |
13 |
345 |
30 |
14 |
+ |
1500 |
62 |
14 |
319 |
24 |
13 |
+ |
5000 |
51 P |
16 P |
233 P |
32 P |
8 P |
Pos controls +S9 |
Name |
2AA |
2AA |
DAN |
BP |
2AA |
Conc. (µg/plate) |
1 |
2 |
10 |
5 |
2 |
|
No. of revertants per plate |
1853 |
351 |
784 |
225 |
329 |
|
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
DAN 1,8-Dihydroxyanthraquinone
P Precipitate
Table 3: Experiment 2 - Without Metabolic Activation
S9 Mix |
Test substance concentration (µg/ plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
– |
0 |
76 |
22 |
353 |
15 |
10 |
– |
50 |
81 |
23 |
368 |
14 |
7 |
– |
150 |
69 |
29 |
373 |
14 |
8 |
– |
500 |
78 |
30 |
351 |
18 |
9 |
– |
1500 |
90 |
30 |
343 |
23 |
8 |
– |
5000 |
94 P |
36 P |
280 P |
24 P |
4 P |
Pos controls |
Name |
ENNG |
ENNG |
MMC |
4NQO |
9AA |
Conc. (µg/plate) |
3 |
5 |
0.5 |
0.2 |
80 |
|
Avg. no. of revertants per plate |
473 |
449 |
998 |
110 |
847 |
|
ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
MMC Mitomycin C
P Precipitate
Table 4: Experiment 2 - With Metabolic Activation
S9 Mix |
Test substance concentration (µg/ plate) |
Number of revertants (mean number of colonies per plate) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA100 |
TA1535 |
TA102 |
TA98 |
TA1537 |
||
+ |
0 |
87 |
15 |
376 |
30 |
12 |
+ |
50 |
75 |
15 |
395 |
23 |
15 |
+ |
150 |
76 |
17 |
384 |
22 |
11 |
+ |
500 |
69 |
17 |
382 |
22 |
9 |
+ |
1500 |
60 |
16 |
338 |
19 |
6 |
+ |
5000 |
54 P |
12 P |
157 P |
21 P |
9 P |
Pos controls +S9 |
Name |
2AA |
2AA |
DAN |
BP |
2AA |
Conc. (µg/plate) |
1 |
2 |
10 |
5 |
2 |
|
No. of revertants per plate |
2950 |
168 |
748 |
152 |
264 |
|
2AA 2-Aminoanthracene
BP Benzo(a)pyrene
DAN 1,8-Dihydroxyanthraquinone
P Precipitate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Bacterial reverse mutation assay
The genotoxic potential of the test item PETMP was assessed in a Bacterial reverse mutation assay according to OECD Guideline 471 and EU method B13/14. Salmonella typhimurium strains TA1535, TA1537, TA102, TA98 and TA100 were treated with solutions of the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate in the first experiment. The experiment was repeated on a separate day using the same dose range as Experiment 1, fresh cultures of the bacterial strains and fresh test material formulations.
The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A white, cloudy precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies.
No significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test material, either with or without metabolic activation.
The test material was considered to be non-mutagenic under the conditions of this test.
Mammalian cell gene mutation assay
The test item PETMP was assessed for its potential to induce mutations at the thymidine kinase locus using the mouse lymphoma cellline L5178Y in accordance with OECD Guideline 476. The se1ection of the concentrations was based on data from the pre-experiment. In experiment I, 600 µg/mL (with metabolic activation) and 65 µg/mL (without metabolic activation) were selected as the highest concentrations. In experiment II, 675 µg/ml (with metabolic activation) and 120 µg/mL (without metabolic activation) were selected as the highest concentrations. Experiment II without metabolic activation was performed as a 24 h long-term exposure assay.
The test item was investigated at the following concentrations:
Experiment I
with metabolic activation:
40, 200,300,400, 450, 500, 550, 600 µg/mL
and without metabolic activation:
10, 20, 30, 40, 50, 55, 60, 65 µg/mL
Experiment II
with metabolic activation:
130,180,230,280,480,530,630,675 µg/mL
andwithoutmetabolic activation:
20,40,50,60,80,100,110, 120 µg/mL
Growth inhibition was observed in experiment land II with and without metabolic activation.
In experiment I with metabolic activation the relative total growth (RTG) was 13.21% for the highest concentration (600 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 65 µg/mL with a RTG of 16.34%. In experiment II with metabolic activation the relative total growth (RTG) was 13.89% for the highest concentration (675 µg/mL) evaluated. The highest concentration evaluated without metabolic activation was 120 µg/mL with a RTG of 10.59%.
In experiment land II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.
Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
The positive controls and showed distinct and biologically relevant effects in mutation frequency.
In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item PETMP is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cellline L5178Y.
Chromosome aberration test
To investigate the potential of PETMP to induce structural chromosome aberrations in Chinese hamster V79 cells, anin vitro chromosome aberration assay was carried out. The chromosomes were prepared 20 h after start of treatment with the test item. The treatment interval was 4 h with and withoutmetabolic activation in experiment I. In experiment II, the treatment interval was 4 h with and
20 h without metabolic activation. Duplicate cultures were treated at each concentration. 100 metaphases per culture were scored for structural chromosomal aberrations.
The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:
Experiment I:
with metabolic activation: 62.5, 125 and 190 µg/ml,
without metabolic activation: 1.95,3.9 and 7.8 µg/ml,
Experiment II:
with metabolic activation: 200, 275 and 300 µg/ml,
without metabolic activation: 0.49, 0.98 and 1.95 µg/ml.
No precipitation of the test item was noted with and without metabolic activation at all concentrations evaluated in experiment l and II.
In experiment I without metabolic activation, toxic effects of the test item were noted at a concentration of 7.8 µg/ml , with metabolic activation at a concentration of 190 µg/ml;
In experiment II without metabolic activation, toxic effects of the test item were observed at a concentration of 1.95 µg/ml, with metabolic activation, toxic effects of the test item were noted at concentrations of 275 µg/ml, and higher.
In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all concentrations treated with the test item were within the historical control data of the negative control.
In experiment I and II with and without metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.
EMS (400 and 600 µg/ml) and CPA (0.83 µg/ml.) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations.
In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item PETMP did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item is considered to be non-clastogenicJustification for classification or non-classification
PETMP was negative, with and without activation, in all required in-vitro tests (gene mutation in bacteria and mammalian cells, in-vitro chromosomal aberration assay) .
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