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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test): The substance DMPEC did not induce mutagenicity in S. typhimurium TA1535, TA97a, TA98, TA100, TA102 in the presence or absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 (OECD 471, GLP)

In vitro cytogenicity (chromosome aberration) study in mammalian cells: the substance DMPEC was concluded to be negative for the induction of chromosome aberrations in the presence and absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation system in human peripheral blood lymphocytes. (OECD 473/GLP).

Gene mutation (mammalian cell gene mutation assay): there was no evidence of induced mutant colonies over background in CHO cells exposed to DMPEC  in the presence or absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation (OECD 476/GLP).

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:
03 Aiugust - 16 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company Ltd.;CP008-170101
- Expiration date of the lot/batch: 30-06-2018
- Purity: 99.7%
Species / strain / cell type:
other: S. typhimurium TA1535, TA97a, TA98, TA100, TA102
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test: 5000, 4000, 3000, 2000, 1000, 500, 250 and 125 µg/plate
Plate incorporation method (Experiment 1): 4000, 1000, 400, 100 and 40 µg/plate
Pre-incubation method (Experiment 2): 2000, 600, 200, 60 and 20 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO

- Justification for choice of solvent/vehicle:Dimethyl Phenylethyl Carbinol found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in any reaction mixture at the concentrations from 2.38 to 0.059 mg/ml corresponding to final test concentrations from 5000 to 125 µg/plate. Hence 5000 µg/plate showing no precipitation was the highest concentration selected for the preliminary cytotoxicity study
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
sodium azide
other: Without S9:3-Methylmethane sulphonate -TA100 and TA102; ICR191 - TA97a With S9: 2-Aminoanthracene - TA1535
Details on test system and experimental conditions:
METHOD OF APPLICATION: iin agar (plate incorporation); preincubation;

DURATION
- Preincubation period: 20 mins
- Expression time: 67 hours for Experiment No. 1 and 72 hours for Experiment No. 2.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn reduction
Evaluation criteria:
1. Criteria for a Positive Response

A test item is considered to be positive (mutagenic), if it induces a concentration dependent increase and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate, in at least one strain with or without metabolic activation system, which is at least 2-fold (3-fold for TA1535) of that observed in the corresponding concurrent vehicle control.

2. Criteria for a Negative Response

A test item for which the results do not meet the above criteria is considered non-mutagenic in this test. In order a substance considered to be negative if the revertant colonies cannot be greater than 2 (or 3 for strain TA 1535), or less than 0.5 in at least 5 doses for all strains tested. However, reproducibility of negative results was confirmed by repeat experimentation.

3. Criteria for an Equivocal Response

Occasionally, a test item cannot be judged to be positive or negative (e.g., concentration dependent increases that fail to reach 2-fold (3-fold for TA1535) control values, or  2-fold (3-fold for TA1535) increases that do not appear to be concentration dependent). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels.
Species / strain:
other: S. typhimurium TA1535, TA97a, TA98, TA100, TA102
Remarks:
Experiment 1
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 examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA1535, TA97a, TA98, TA100, TA102
Remarks:
Experiment 2
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:
not examined
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: DMPEC found to be miscible in DMSO at a concentration of 50 mg/ml. No precipitation was observed in any reaction mixture at the concentrations from 2.38 to 0.059 mg/ml corresponding to final test concentrations from 5000 to 125 µg/plate (Appendix 4).

RANGE-FINDING/SCREENING STUDIES:
Before commencing the study, the test item was assessed for cytotoxicity to the tester bacteria using tester strain TA100. Eight concentrations (5000, 4000, 3000, 2000, 1000, 500, 250 and 125 µg/plate) of the test item, formulated in dimethyl sulphoxide, were tested for toxicity to bacterial cells.

Plate Incorporation Method
Slight cytotoxicity was observed to the bacterial background lawn at the concentrations of 5000 and 4000 µg/plate, in the absence of metabolic activation. Moderate cytotoxicity was observed to the bacterial background lawn at the concentration of 5000 µg/plate, whereas slight cytotoxicity was observed at 4000 µg/plate in the presence of metabolic activation. Hence, 4000 µg/plate was selected as the maximum test concentration for the main study in both the presence and absence of metabolic activation.

Pre Incubation Method
Severe cytotoxicity was observed to the bacterial background lawn at concentrations of 3000 to 5000 µg/plate, slight cytotoxicity was observed at the concentration of 2000 µg/plate in the absence of metabolic activation. Severe cytotoxicity was observed to the bacterial background lawn at concentrations of 5000 and 4000 µg/plate, significant (very thin lawn) cytotoxicity was observed at the concentration of 3000 µg/plate and no cytotoxicity was observed from 2000 to 125 µg/plate in the presence of metabolic activation. Hence 2000 µg/plate was selected as the maximum test concentration for the main study in both the presence and absence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: Concurrent positive controls demonstrated sensitivity of the assay with and without metabolic activation (Appendix 6).

- Negative (solvent/vehicle) historical control data: Plate counts for the spontaneous histidine revertant colonies in the vehicle control groups were found to be within the frequency ranges expected from the laboratory historical control data at INTOX PVT. LTD (Appendix 6).
Conclusions:
Under the conditions described for this study, it is concluded that DMPEC is non-mutagenic in Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the presence and absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria (16937), strains of S. typhimurium TA1535, TA97a, TA98, TA100, TA102 were exposed to DMPEC (99.7%) in DMSO at concentrations of 4000, 1000, 400, 100, 40 and 0 μg/plate (direct plate incorporation; experiment 1) and 2000, 600, 200, 60, 20 and 0 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9).

DMPEC was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 September 2017 - 28 February 2018
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)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company Ltd.; CP008-170101
- Expiration date of the lot/batch: 30-06-2018
- Purity: 99.7%

Species / strain / cell type:
other: Human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Healthy adult male
- Cell cycle length, doubling time or proliferation index: 12-16 hours
- Modal number of chromosomes: 46

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI medium 1640; ~5% CO2 in air, 90 to 100% humidity
- Properly maintained: Yes

Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 2000, 1000, 500, 250 and 125 μg/ml
Main tests (with and without S9): 100, 300, 1000 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hrs with and without S9; 24 hrs without S9
- Expression time (cells in growth medium): 24 hrs

SPINDLE INHIBITOR (cytogenetic assays): Colchicine, final concentration 0.5 µg/ml

STAIN (for cytogenetic assays): 5% Giemsa (v/v)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Each culture was harvested and processed separately. At the end of the incubation period each culture was transferred to a centrifuge tube. The cell suspension was centrifuged at about 300 rpm for 5 minutes and the supernatant was discarded. To each of these tubes 5 ml of 0.56% potassium chloride (KCl) was added for hypotonic treatment of the cells at room temperature for 20 minutes. After hypotonic treatment, the suspension was once again centrifuged at about 1200 rpm for 5 minutes and supernatant discarded. The freshly obtained pellet was fixed by re-suspending in 5 ml fresh cold methanol:acetic acid (3:1) and left at room temperature for 10 minutes. Then it was centrifuged at about 1200 rpm for 5 minutes and the supernatant was discarded. After the third change of fixative the pellet was re-suspended in about 2 ml of fixative and the solution was dropped on to clean chilled glass microscope slides.
The chromosome preparations were heat fixed and later stained with 5% Giemsa for 10 minutes. The slides were air dried, mounted with DPX, coded and examined for well spread metaphases under microscope with 100X objective.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least 300 metaphases were evaluated per test concentration and for the vehicle and positive controls, equally divided among the duplicates

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
The chromosome aberrations were divided into three main groups:
- Chromatid type,
- Chromosome type,
- Other types of aberrations.

Under the chromatid and chromosome type aberrations, gaps, breaks or fragments and exchanges were scored and recorded. Other types of abnormalities such as multiple chromatid breaks, pulverisations, deletions, ring chromosomes, polyploidy were scored and recorded. For the purpose of calculation of frequency of chromatid and chromosome type aberrations, `gaps’ were not considered as structural chromosome abnormalities.
Evaluation criteria:
1. Criteria for a Positive Response
A test item is considered as mutagenic if it produces a concentration related increase or a reproducible increase at one or more concentrations in number of cells with chromosome aberrations with or without metabolic activation system, which is statistically significant.

2. Criteria for a Negative Response
A test item is considered non-mutagenic, if it does not induce any increase in the number of cells with chromosome aberrations, and does not show any dose-response relationship, either with or without metabolic activation.

3. Criteria for an Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative (e.g., statistically significant increases in number of cells with chromosome aberrations with or without metabolic activation system that do not appear to be concentration dependent, or increase in single replicate of culture at one or more concentrations). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified study plan such as appropriate spacing of dose levels and the metabolic activation conditions.
Statistics:
The data from each treatment level and positive control substance are to be compared with the solvent control using a Chi-square test (2*2 contingency tests). Statistical analyses and Poison-based 95% control limits for the distribution of historical negative control data will be used for assessment of the statistical significance. All analyses and comparisons are evaluated at 5% (p = 0.05) level.
Species / strain:
other: human peripheral blood lymphocytes
Remarks:
3 hours
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: human peripheral blood lymphocytes
Remarks:
24 hrs
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item, Dimethyl Phenylethyl Carbinol, found to be miscible in DMSO. Fresh preparations were employed for the test. The test formulation in a volume of 0.2 ml was added to the tubes containing final reaction mixture (containing 9.6 ml culture medium, 0.4 ml whole blood without or with the metabolic activation system (S9), in a volume of 0.5 ml). The tubes were examined with unaided eyes, for evidence of complete solubility of the test item or formation of a precipitate. Dimethyl Phenylethyl Carbinol revealed heavy precipitation in the final reaction mixture tubes at 5000 μg/ml to 3000 μg/ml test concentration, while slight precipitation was observed at 2000 μg/ml test concentration and no precipitation was observed at 1000 μg/ml to 125 μg/ml test concentration. Thus 2000 μg/ml was selected as the highest concentration for preliminary cytotoxicity test.

RANGE-FINDING/SCREENING STUDIES:
Experiment No. 1 (3 hours treatment without metabolic activation)
Severe cytotoxic reduction in mitotic index over vehicle control was observed at the test concentration of 2000 μg/ml. The slight reduction in mitotic indices of highest treated group over vehicle control was 46.11% at the concentration of 1000 μg/ml. The values of % reduction in mitotic indices of treated group over vehicle control at the concentrations of 2000, 1000, 500, 250 and 125 μg/ml were 52%, 46.11%, 34%, 28.57% and 16% respectively. Hence for the main experiment 1000 μg/ml showing slight cytotoxicity was selected as highest concentration with lower concentrations of 300 and 100 μg/ml.

Experiment No. 2 (24 hours treatment without metabolic activation)
Severe cytotoxic reduction in mitotic index over vehicle control was observed at the test concentration of 2000 μg/ml. The slight reduction in mitotic indices of treated group over vehicle control was 43.14% at the concentration of 1000 μg/ml. The values of % reduction in mitotic indices of treated group over vehicle control at the concentrations of 2000, 1000, 500, 250 and 125 μg/ml were 51.47%, 43.14%, 33.33%, 25.49% and 7.84% respectively.
Hence for the main experiment 1000 μg/ml showing slight cytotoxicity was selected as highest concentration with lower concentrations of 300 and 100 μg/ml.

Experiment No. 3 (3 hours treatment with metabolic activation)
Severe cytotoxic reduction in mitotic index over vehicle control was observed at the test concentration of 2000 μg/ml. The slight reduction in mitotic indices of treated group over vehicle control was 42.09% at the concentration of 1000 μg/ml. The values of % reduction in mitotic indices of treated group over vehicle control at the concentrations of 2000, 1000, 500, 250 and 125 μg/ml were 51.04%, 42.09%, 30.19%, 22.64% and 18.87% respectively.
Hence for the main experiment 1000 μg/ml showing slight cytotoxicity was selected as highest concentration with lower concentrations of 300 and 100 μg/ml.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation)
- Positive historical control data: Yes – 2017 data (Appendix 6)
- Negative (solvent/vehicle) historical control data: Yes – 2017 data (Appendix 6)
Conclusions:
In an in vitro cytogenicity (chromosome aberration) study in human peripheral blood lymphocytes, DMPEC did not induce any significant and/or concentration related increases in the incidence of chromosome aberrations either in absence or presence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation system.
Executive summary:

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (16938), human peripheral blood lymphocytes were exposed to DMPEC (99.7%) in DMSO at concentrations of 100, 300 and 1000 μg/mL in the absence or presence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation system.

Cytotoxicity was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Positive controls induced the appropriate response. There was no evidence of a statistically significant increase in chromosomal aberrations with or without metabolic activation.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline [In vitro mammalian cytogenetics [Chromosome aberration]] OECD 473 for in vitro cytogenetic mutagenicity data

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)
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
- Source and lot/batch No.of test material: Yinghai (Cangzhou) Aroma Chemical Company Ltd.; CP008-170101
- Expiration date of the lot/batch: 30-06-2018
- Purity: 99.7%

Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Centre for Cell Sciences, Pune, India.
- Methods for maintenance in cell culture if applicable: The cultures in petriplates during cloning were maintained in Ham's F-12 Nutrient Mixture (F-12) with 10% (v/v) of heat-inactivated fetal bovine serum while cells during selection were maintained in the same medium but containing 6-thioguanine as a selective agent.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham’s F-12K with L-glutamine containing 10% FBS and Penicillin-Streptomycin 1X; ~ 5% CO2 in air, 85 to 95% humidity.
- Periodically checked for Mycoplasma contamination: Supplied as microbial free
- Properly maintained: Yes
- Periodically 'cleansed' against high spontaneous background: Yes
Metabolic activation:
with and without
Metabolic activation system:
Sodium phenobarbitone and β- naphthoflavone-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test: 1250, 625, 312.5, 156.25 and 78.125 μg/ml
Main test (with and without S9): 1250, 625, 312.5 and 156.25 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 3 hrs with and without S9
- Expression time (cells in growth medium): 8 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative survival

Evaluation criteria:
The criteria for determining a positive, negative and equivocal results are listed below. However observations on borderlines of these criteria were verified at the discretion of the study director.
1. Criteria for a Positive Response
A test item is considered as mutagenic if, treatment with the test item produces a dose dependent increase in the mutant frequencies, where in the elevation of mutant frequencies above 40 mutants/106 clonable cells, is considered to be a positive response.
2. Criteria for a Negative Response
A test substance for which the results do not meet the above criteria is considered non- mutagenic in this test.
3. Criteria for Equivocal Response
Occasionally, a test item cannot be judged to be positive or negative (e.g. > 40 mutants/106 clonable cells that do not appear to be concentration dependent). In these rare instances, the results may be classified as equivocal. Equivocal or weak positive results may indicate the need to repeat the test, possibly with a modified Study Plan such as appropriate spacing of dose level.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1250 μg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and pH: Concentrations from 312.5 – 5000 μg/ml were checked for pH and precipitation. Heavy precipitation was observed at the concentrations of 500 and 250 mg/ml. Slight precipitation was observed at the concentration of 125 mg/ml. The pH was found to be within the desired pH range (7.0 ± 1).


RANGE-FINDING/SCREENING STUDIES:
For assessing cytotoxicity of the test item, CHO cells were exposed to five concentrations of Dimethyl Phenylethyl Carbinol viz. 1250, 625, 312.5, 156.25 and 78.125 μg/ml in treatment medium. Cytotoxicity was determined by relative survival (RS) following about 3 h treatments, in presence as well as in absence of a metabolic activation system (S9). Slight cytotoxicity was observed at the concentration of 1250 μg/ml, no cytotoxicity was observed at the concentrations from 625 to 78.125 μg/ml. However the relative survival (RS) was observed to be 19.4, 72.4, 73.7, 79.0 and 84.2% in absence of S9 and 19.1, 71.3, 74.3, 80.9 and 83.3% in presence of S9 at the concentrations from 1250 to 78.125 μg/ml respectively. The concentration of 1250 μg/ml was selected as the highest concentration in presence and absence of metabolic activation system, for the definitive study.
Conclusions:
In an in vitro gene mutation study (hprt) in CHO cells, DMPEC was not mutagenic in the presence or absence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation.
Executive summary:

In an in vitro gene mutation study in mammalian cells (16939), Chinese hamster ovary cells were exposed to DMPEC (99.7%) in DMSO at concentrations of 1250, 625, 312.5 and 156.25 μg/ml for 3 hrs with and without sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation.

DMPEC was tested up to the limits of cytotoxicity. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 476 for in vitro cytogenetic gene mutation data.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation (Bacterial Reverse Mutation Assay/Ames test)

There is one in vitro gene mutation study (reverse gene mutation assay/Ames test in bacteria) available.

In a reverse gene mutation assay in bacteria (OECD 471/GLP), strains of S. typhimurium TA1535, TA97a, TA98, TA100, TA102 were exposed to DMPEC (99.7%) in DMSO at concentrations of 4000, 1000, 400, 100, 40 and 0 μg/plate (direct plate incorporation; experiment 1) and 2000, 600, 200, 60, 20 and 0 μg/plate (20 minute pre-incubation; experiment 2) in the presence and absence of mammalian metabolic activation (Sodium phenobarbitone and β-naphthoflavone-induced rat liver S9). DMPEC was tested up to the limit concentration (5000 µg/plate). The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

In vitro cytogenicity (chromosome aberration) study in mammalian cells:

There is one in vitro cytogenicity (chromosome aberration) study in mammalian cells available.

In an in vitro cytogenicity (chromosome aberration) study in mammalian cells (OECD 473/GLP), human peripheral blood lymphocytes were exposed to DMPEC (99.7%) in DMSO at concentrations of 100, 300 and 1000 μg/mL in the absence or presence of sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation system. Cytotoxicity was satisfactory to provide adequate metaphase cells for examination of chromosomal aberrations. Positive controls induced the appropriate response. There was no evidence of a statistically significant increase in chromosomal aberrations with or without metabolic activation.

Gene mutation (mammalian cell gene mutation assay):

There is one gene mutation (mammalian cell gene mutation assay) available.

In an in vitro gene mutation study in mammalian cells (OECD 476/GLP), Chinese hamster ovary cells were exposed to DMPEC (99.7%) in DMSO at concentrations of 1250, 625, 312.5 and 156.25 μg/ml for 3 hrs with and without sodium phenobarbitone and β-naphthoflavone-induced rat liver S9 metabolic activation. DMPEC was tested up to the limits of cytotoxicity. The positive controls induced the appropriate response. There was no evidence of induced mutant colonies over background.

All 3 studies are clearly negative and are suitable to use in the human health hazard assessment.

Justification for classification or non-classification

Based on the available information in the dossier, the substance DMPEC (CAS No. 103-05-9) does not need to be classified for germ cell mutagenicity when the criteria outlined in Annex I of 1272/2008/EC are applied.