Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The available data indicate that 2-phenoxyethanol is not genotoxic.
Negative in the Ames test, negative results in mammalian chromosomal aberration and gene mutation tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Sep 2001 - 21 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan 287 (Japan EPA), Eisei 127 (MHW) and Heisei 09/10/31 Kikyoku No. 2 (MITI)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden
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): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: 664287
- Stability under test conditions: The stability of the test substance in water over a period of 96 hours has been verified analytically by the data owner.
- Storage condition of test material: Room temperature, under nitrogen
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 (minimal essential medium) supplemented with 10% FCS (fetal calf serum)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 mix. The S9 was prepared from 8-12 weeks old male Wistar Hanlbm rats induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days. The livers were prepared 24 hours after the last treatment.
Test concentrations with justification for top dose:
Pre-test: 10.9, 21.9, 43.8, 87.5, 175, 350, 700 and 1400 µg/mL
Experiment I: 43.8, 87.5, 175, 350, 700 and 1400 µg/mL
Experiment II and III: 175, 350, 525, 700, 1050 and 1400 µg/mL
The highest applied concentration in the pre-test on toxicity was chosen with regard to the molecular weight of the test substance with respect to the OECD guideline.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: without S9 mix: 4 and 18 hours; with S9 mix: 4 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: In each chamber, 1 x 10^4 to 6 x 10^4 cells were seeded (at least 2 chambers per dish and group). 100 metaphase plates per culture were scored for structural chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: The number of polyploid cells was determined of each test group in a sample of 500 cells per culture (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).
Evaluation criteria:
A test substance is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of historical control data (0.0 - 4.0% aberrant cells exclusive gaps).
A test substance is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of historical control data (0.0 - 4.0% aberrant cells exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations are not in the range of historical control data (0.0% - 8.5% polyploid cells)
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
ambiguous
Remarks:
see remarks on results
Cytotoxicity / choice of top concentrations:
other: In the pre-test on toxicity cell numbers 24 hours after start of treatment were scored for cytotoxicity. Distinctly decreased cell numbers were scored with 700 µg/mL and above.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No clear toxic effects were observed after 4 hours treatment up to the highest evaluated test substance concentration (1400 µg/mL).

Genotoxicity without metabolic activation:

A single statistically significant increase (p<0.05) of aberrant cells was observed in experiment I after 4 hours treatment with 1400 µg/mL. Although this increase of 4% aberrant cells, exclusive gaps, was statistically significant compared to the low response in the corresponding solvent control (1% aberrant cells, exclusive gaps), the value is within the historical control data range. Therefore, the statistical significance is regarded as being biologically irrelevant.

For tables of results on chromosomal analyses refer to attached background material.

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep 2001 - 5 Dec 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesanstalt für Pflanzenbau und Pflanzenschutz Rheinland-Pfalz, Mainz
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): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Purity test date: 18 Sep 2001
- Lot/batch No.: 66-4287
- Stability under test conditions: The stability of the test substance throughout the study period (in the vehicle DMSO over 4 hours and in water over 96 hours) was verified by reanalysis.
- Storage condition of test material: Room temperature (N2 conditions)
- Test Substance No.: 01/0498-1
Target gene:
his-operon (S. typhimurium)
trp-operon (E. coli)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix. At least 5 male Sprague-Dawley rats received a single intraperitoneal injection of 500 mg Aroclor 1254 per kg body weight. After 5 days, rats were sacrificed, the livers were prepared, and the S-9 fraction was obtained by standard methods.
Test concentrations with justification for top dose:
0, 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: With S-9 mix: 2-aminoanthracene (2-AA); without S-9 mix: N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48-72 hours in the dark

NUMBER OF REPLICATIONS: 3 culture plates per strain per dose or per control were prepared.

NUMBER OF CELLS EVALUATED: Not applicable, as the number of revertant colonies was calculated.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
not applicable
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No bacteriotoxic effect was observed in the standard plate test. In the preincubation assay a slight decrease in the number of revertants and/or slight reduction in the titer was occasionally observed depending on the strain and test conditions.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No bacteriotoxic effect was observed in the standard plate test. In the preincubation assay a slight decrease in the number of revertants and/or slight reduction in the titer was occasionally observed depending on the strain and test conditions.

Refer to attached background material

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Sep 2001 - 26 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan 287 (Japan EPA), Eisei 127 (MHW) and Heisei 09/10/31 Kikyoku No. 2 (MITI)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden
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): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: 664287
- Stability under test conditions: The stability of the test substance in water over a period of 96 hours has been verified analytically by the data owner.
- Storage condition of test material: Room temperature, under nitrogen
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) supplemented with 10% FCS (fetal calf serum)
- 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 mix. The S9 was prepared from 8-12 weeks old male Wistar Hanlbm rats induced by applications of 80 mg/kg b.w. Phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days. The livers were prepared 24 hours after the last treatment.
Test concentrations with justification for top dose:
Experiment I and II, with and without S9 mix: 43.8, 87.5, 175, 350, 700 and 1400 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 days

SELECTION AGENT (mutation assays): 6-TG (6-thioguanine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Approximately 1.5 x 106 (single culture) and 5 x 102 cells (in duplicate) were seeded for the determination of mutation rate and toxicity, respectively. Stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test substance is classified as positive if it induces a concentration-related increase of the mutant frequency.
A test substance producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test substance is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment and if this increase exceeds the range of historical negative or solvent control data.
The test substance is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.5 - 33.1 mutants per 10^6 cells) a concentration-related increase of the mutations within this range has to be discussed.
Statistics:
Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
ambiguous
Remarks:
See remarks on results
Cytotoxicity / choice of top concentrations:
no cytotoxicity

Genotoxicity without metabolic activation:

An increase of the induction factor exceeding the threshold of three times the corresponding solvent control occurred in the second culture of experiment I without metabolic activation at 350 µg/mL. However, this increase was judged to be based upon the rather low solvent controls of 6.0 and 3.6 mutant colonies/10^6 cells. Compared to the corresponding negative control the threshold was not reached.

Genotoxicity with metabolic activation:

An increase of the induction factor exceeding the threshold of three times the corresponding solvent control occurred in the second culture of experiment I at 1400 µg/mL in the presence of metabolic activation. However, this increase was judged to be based upon the rather low solvent controls of 6.0 and 3.6 mutant colonies/10^6 cells. Compared to the corresponding negative control the threshold was not reached. The historical range of negative and solvent controls was only exceeded in the first culture of the second experiment at 87.5 and 175 µg/mL with metabolic activation. Although this increase exceeded the historical range of solvent controls, it was not dose-related and no comparable effect occurred in the parallel culture under identical conditions or in the first experiment. Therefore, the isolated effect was judged an artefact.

For tables of results refer to attached background material.

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jul 1994 - 19 Sep 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
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): MARLOPHEN P 1, 2-Phenoxyethanol
- Physical state: liquid, clear, brownish
- Analytical purity: 85.7% Phenoxyethanol
- Impurities (identity and concentrations): 12% Phenoxydiethanol (GC-Analysis)
- Purity test date: September 1993
- Lot/batch No.: P 86, UB 271
- Other: Date of manufacture: 06 Sep 1993; ID No.: 0637/81537; Sample No. of testing laboratory: 1432/931008
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium, other: TA 1538, TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Arochlor-activated S9 fraction from Cytotest Cell Research GmbH und Co. KG (Lot No.: 060694 and 170593). Enzyme activity was tested in all strains using anthracene.
Test concentrations with justification for top dose:
8, 40, 200, 1000, 5000 µg/plate
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:
9-aminoacridine
2-nitrofluorene
sodium azide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 min
- Exposure duration: 96 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A substance which at 5000 µg/plate still shows no induction of mutagenic effects can be regarded as not mutagenic (Ames, B.N. et al.: Mutation Research, 31 6, 347-364 (1975)).
Statistics:
The reported standard deviations, mean values, and factors were calculated using a BIOSYS software, according to basic mathematical calculations.
Species / strain:
S. typhimurium, other: TA 1538, TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In both the main and the preincubation test, the test substance MARLOPHEN P 1 proved to be non-mutagenic up to 5000 µg/plate in the presence and absence of arochlor-induced liver microsomes in all tested strains.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
No toxicity effects were observed throughout the tests.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

For tables of results refer to attached background material.

Conclusions:
Interpretation of results:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The available data indicate that 2-phenoxyethanol is not genotoxic.
Negative in chromosomal aberration, micronucleus and unscheduled DNA synthesis assays.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 Oct 2001 - 12 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The initial body weights of 4 males were slightly over 45 g (45.8-47.8 g). The historical data used in this study is the summary of the data obtained in the years 1999 and 2000. These deviations, however, do not affect the validity of the study.
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan 287 (Japan EPA), Eisei 127 (MHW) and Heisei 09/10/31 Kikyoku No. 2 (MITI)
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden
Type of assay:
micronucleus assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: 664287
- Stability under test conditions: The stability of the test substance in water over a period of 96 hours has been verified analytically by the data owner.
- Storage condition of test material: Room temperature, under nitrogen
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 8 to 10 weeks at start of acclimatization
- Weight at study initiation: 40.7 ± 4.1 g at start of treatment. The initial body weights of 4 males were slightly over 45 g (45.8 - 47.8 g)
- Assigned to test groups randomly: yes
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 4
- Humidity (%): 30 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: In accordance with the scheduled dose to be tested.
Duration of treatment / exposure:
not applicable
Frequency of treatment:
once
Post exposure period:
24 and 48 h after treatment
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
10 mL/kg body weight total volume applied
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
10 mL/kg body weight total volume applied
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
10 mL/kg body weight total volume applied
No. of animals per sex per dose:
6 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: intraperitoneally, once
- Doses / concentrations: 40 mg/kg b.w.
Tissues and cell types examined:
Tissue: Bone marrow
Cell type: erythrocytes in bone marrow
Evaluation criteria:
A test substance is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes, which clearly exceeds the negative control range or a relevant positive response for at least one of the test points.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system.
Statistics:
The statistical significance of the findings at p<0.05 was assessed by means of the non-parametric Mann-Whitney test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In the main experiment the males treated with the highest dose (500 mg/kg) showed clear signs of toxicity. For details refer to attached background material.

For tables of results refer to attached background material.

Conclusions:
Interpretation of results: negative
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Sep 2001 - 12 Feb 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan 287 (Japan EPA), Eisei 127 (MHW) and Heisei 09/10/31 Kikyoku No. 2 (MITI)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Landwirtschaft und Forsten, Wiesbaden
Type of assay:
unscheduled DNA synthesis
Specific details on test material used for the study:
- Name of test material (as cited in study report): Protectol PE
- Physical state: colorless liquid
- Analytical purity: 99.9%
- Lot/batch No.: 664287
- Stability under test conditions: The stability of the test substance in water over a period of 96 hours has been verified analytically by the data owner.
- Storage condition of test material: Room temperature, under nitrogen
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, Füllinsdorf, Switzerland
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 219.8 g (SD ± 17.8 g)
- Assigned to test groups randomly: yes
- Fasting period before study: The animals were starved overnight (2 hours treatment) or approximately 6 hours (16 hours treatment).
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)
- Concentration of test material in vehicle: In accordance with the scheduled dose to be tested
- Amount of vehicle (if gavage or dermal): 10 mL/kg body weight
Duration of treatment / exposure:
not applicable
Frequency of treatment:
once
Post exposure period:
2 and 16 hours after treatment
Dose / conc.:
875 mg/kg bw (total dose)
Remarks:
10 mL/kg b.w. total volume applied
Dose / conc.:
1 750 mg/kg bw (total dose)
Remarks:
10 mL/kg b.w. total volume applied
No. of animals per sex per dose:
4 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
For 2 hours preparation interval: N,N’-dimethylhydrazinedihydrochloride (DMH)
- Route of administration: orally, once
- Doses / concentrations: 40 mg/kg b.w.

For 16 hours preparation interval: 2-acetylaminofluorene
- Route of administration: orally, once
- Doses / concentrations: 100 mg/kg b.w.
Tissues and cell types examined:
Cell types examined: isolated primary hepatocytes
Evaluation criteria:
Nunclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test substance is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.
A test substance producing net grains no greater than 0 at anyone of the test points is considered non-effective in this system.
Statistics:
Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see remarks on results

Toxicity:

In the pre-experiment, the following symptoms occurred: reduction of spontaneous activity, abdominal position, eyelid closure, apathy, ruffled fur (after 1-6, 24 and 48 hours). The symptoms abdominal position, eyelid closure and apathy were no longer noted after 48 hours. On the basis of the results of the pre-experiment, 1750 mg/kg b.w. was estimated to be suitable as maximum tolerable dose for the main experiment.

In the main experiment, high mortality rates were reported at the 16 preparation interval at the test dose of 1750 mg/kg b.w.; therefore, additional animals were used. From the 10 animals used, 5 died. No further clinical signs were reported.

The viability of the hepatocytes was not substantially affected due to the in vivo treatment with the test substance. The interindividual variations obtained for the numbers and the viabilities of the isolated hepatocytes are in the range of the historical laboratory control.

For table of results on UDS test refer to attached background material.

Conclusions:
Interpretation of results: negative
Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 1987 - 04 Feb 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
regarding test report
Principles of method if other than guideline:
No guideline mentioned in the report.
However, study is equivalent or similar to OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test) with deviations regarding test report:
- Weight of the animals is not reported.
- No historical negative control data are presented.
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): 2-Phenoxyethanol, PHENOXETOL
- Physical state: colorless liquid
- Analytical purity: 99.83%
- Lot/batch No.: 53
- Other: Source: NIPA Laboratories, Ltd.
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Portage, MI
- Age at study initiation: 8-11 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002 (Ralston Purina Company Richmond, IN), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 40-60
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: Target concentrations: 0, 28.0, 93.3, 280 mg/ml; observed concentrations: ND, 36.5 ± 1.2, 88.1 ± 2.1, 368.7 ± 10.5 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Duration of treatment / exposure:
6, 24, 48 h
Frequency of treatment:
once
Post exposure period:
none
Dose / conc.:
280 mg/kg bw (total dose)
Dose / conc.:
933 mg/kg bw (total dose)
Dose / conc.:
2 800 mg/kg bw (total dose)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Trimethylphosphate
- Route of administration: oral: gavage
- Doses / concentrations: 2000 mg/kg bw
Tissues and cell types examined:
bone marrow
Evaluation criteria:
The test material was considered positive if it induced a significant and dose-related increase in total aberration frequency (chromatid-type plus chromosome-type aberrations). Significant increases in the total aberration frequency at only a single dose level was to be verified with an additional experiment. The test chemical was considered negative if it failed to induce a significant increase in the total aberration frequency at the 2 high dose levels (provided there was not excessive cell toxicity). Significant increases in miscellaneous aberrations and severely damaged cells (if present) were evaluated critically for biological significance.
Statistics:
The following parameters were evaluated for statistical significance: total aberrations (excluding gaps), number of cells with aberrations (excluding
gaps), number of gaps, number of cells with gaps, miscellaneous aberrations (e.g. chromosomal disintegration and pulveration), and severely damaged cells. The raw data were first transformed by adding 1 to each count and then taking the natural log of the adjusted number. The transformed data and data on mitotic indices were analyzed by a three-way analysis of variance (sex, dose and time), assuming the three-way interaction to be zero. Depending on a review of two-way interactions, the data for main effects were analyzed by one, two, or three-way analysis of variance. Pairwise comparisons of treated vs. negative control data were made using a t-test (with Bonferroni correction for multiple comparisons). The criterion for significance was p < 0.01.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
see remarks on results

The LD50 value (moving average method) was 2937 and 4013 mg/kg bw for males and females. Based on these results, 2800 mg/kg bw was chosen as the highest dose to be used in the cytogenicity study. GC analysis revealed that the concentrations of test material in the low and high dose groups were higher than the targeted concentrations. Food consumption in the high dose and positive control groups was lower than the negative control. Cytogenetic data from one animal each in the control and high dose group could not be collected at 6 hrs due to technical errors. One male in the high dose group died before the scheduled euthanization time of 6 and 24 hours, and one negative control male and 3 high dose males did not survive to the scheduled euthanization time of 48 hours. Among females treated with 2800 mg/kg bw, 1, 3, and 3 animals died before the scheduled euthanization times of 6, 24 and 48 hours, respectively. All positive controls and animals treated with lower doses of test material survived. There were no significant increases in the incidence of total or specific type of cytogenetic anomalies in groups treated with 933 or 2800 mg/kg bw test material at any time point compared to negative controls. The highest number of aberrations observed in treated animals was 6 (in female rats treated with 933 mg/kg bw for 6 hrs) compared to 3 in female controls at the same time point. There was no effect of treatment on the percentage of cells with gaps. Data on mitotic indices indicated that there was no excessive cell toxicity among groups treated with 933 and 2800 mg/kg bw. The positive controls induced a significant increase in the total number of aberrations (48 and 54 in males and females, respectively). Therefore, the test was valid.

For tables of results refer to attached background material.

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genotoxicity:

2 -Phenoxyethanol was tested for genotoxic potential in an adequate battery of in vitro and in vivo tests with various end points.

In vitro: 2-Phenoxyethanol was not a point mutagen in studies on bacteria at concentrations up to 5000 µg/plate with and without metabolic activation (BASF AG, 2002; Sasol, 1994; Nipa Laboratories, 1982). Further tests on point mutations on the HGPRT locus in eukaryotic cells yielded also negative results (BASF AG, 2002, The Dow Chemical Company, 1987).
In vitro testing on chromosome-damaging effects in Chinese hamster cell cultures indicated no effects with and without metabolic activation (BASF AG, 2002; Unilever, 1985).

The available data indicate that 2 -phenoxyethanol was neither an in vitro cell mutagen nor a clastogen.

In vivo: The in vivo assays also showed no mutagenic effects with 2-phenoxyethanol treatment.
No chromosome-damaging effects were observed and testing on DNA damage in vivo via the UDS test in Wistar rat also failed to show mutagenic effects. (BASF, 2002, Nipa Laboratories, 1982; BASF AG, 2002; The Dow Chemical Company, 1988)

The available data indicate that 2-phenoxyethanol was not an in vivo cell mutagen or clastogen.

Overall, 2-phenoxyethanol is unlikely to pose a genotoxic hazard to man.

Justification for classification or non-classification

The available data are conclusive but not sufficient for classification.