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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay (Ames / OECD guideline 471): negative

In vitro mammalian chromosomal aberration assay (ChrAb / OECD guideline 473): negative

In vitro mammalian cell gene mutation assay (MLA / OECD guideline 476): negative

DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro (OECD guideline 482): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Reason / purpose for cross-reference:
reference to same study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Neither E.coli WP2 strains nor S. typhimurium TA102 were used.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
1. test: 4, 20, 100, 500, 2500 µg/plate; solvent: acetone (experiment failed due to toxic effects of the solvent)
2. test: 4, 20, 100, 500, 2500 µg/plate; solvent: DMSO
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
untreated cells
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: +S9: 2-aminoanthracene; -S9: sodium azide (TA100, 1535), 4-nitro-o-phenylendiamine (TA98, 1537, 1538)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
Evaluation criteria:
According to Guideline.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA, 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
interpretatin of results: negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 AUG - 30 SEP 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Neither E.coli WP2 strains nor S. typhimurium TA102 were used.
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
Neither E.coli WP2 strains nor S. typhimurium TA102 were used.
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 applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction
Test concentrations with justification for top dose:
without metabolic activation: 0, 1.5, 5, 15, 50, 150 and 500 µg/plate
with metabolic activation: 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-ethyl-N'-nitrosoguanidine (3-5 µg/plate), 9-aminoacridine (80 µg/plate), 4-Nitro-o-phenylenediamine (5 µg/plate), 4-nitroquinoline-N-oxide (0.2 µg/plate), 2-aminoanthracene (0.5-2 µg/plate)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A known aliquot (0.5 mL) of S9-mix and 2 mL of molten, trace histidine supplemented media were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix.

Incubation and Assessment of Plates: all of the plates were incubated at 37 ºC for approximately 48 h and the frequency of revertant colonies assessed using a Domino colony counter.

NUMBER OF REPLICATIONS: Triplicate

DETERMINATION OF CYTOTOXICITY
- Method: The cytotoxicity was rated as follows:
S= sparse background lawn
V= very thin background lawn
T= toxic
NT= not tested at this dose level
Evaluation criteria:
The test item was considered positive if:
There is a dose related increase in the number of revertants or a biologically relevant increase for at least one test concentration.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Number of Revertants (Number of Colonies per plate)
Base-pair Substitution Type Frameshift Type
TA100 TA1535 TA1538 TA98 TA1537
147 33 29 29 9
124    (133) 32    (34) 21  (25) 40   (31) 7   (10)
129 36 24 33 13
2225 (22)b19
Number of Revertants (Number of Colonies per plate)
Base-pair Substitution Type Frameshift Type
TA100 TA1535 TA1538 TA98 TA1537
88 15 9 23 7
93    (88) 19    (16) 7   (9) 22   (22) 8   (9)
84 15 10 21 12
Conclusions:
interpretation of results: negative

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the test material using the Ames plate incorporation method in triplicates, 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 ranged between 1.5 and 5000 µg/plate in the first experiment. The vehicle (dimethyl sulphoxide) control plates produced counts of revertant colonies within the normal range. All the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without the metabolising system. The test material caused a visible reduction in the growth of the bacterial lawn to all of the strains of salmonella tested. The sensitivity of the bacterial strains to the toxicity of the test material varied between strains and between exposures with or without S9 -mix. The first incidence of toxicity was recorded at 150µg/plate. The test material was, therefore, tested up to its toxic limit. The test material was considered to be NON-MUTAGENIC under the conditions of this test.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study similar to OECD Guideline.
Qualifier:
according to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Species / strain / cell type:
hepatocytes: The liver of a male Fischer 344 rat was perfused, excised and combed yielding 13.2 x 10E5 hepatocytes/mL of medium with 100% viability.
Details on mammalian cell type (if applicable):
- Type and identity of media: WME containing 10% calf serum
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Metabolic activation system:
Not applicable; primary hepatocytes are metabolic competent
Test concentrations with justification for top dose:
Pre-Test: 0.5, 5, 25, 50, 100, 500, 750, 1000, 2500, 5000 µg/mL and 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25, 50, 100 µg/mL
Main study: 0.25, 0.5, 1.0 and 2.5 µg/mL
Vehicle / solvent:
- Vehicle used: DMSO
Untreated negative controls:
yes
Remarks:
untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Scheduled DNA "repair" synthesis, evidenced by a net increase in black silver grains over the nucleus, is quantified by determining nuclear and cytoplasmic grain counts using an automated colony counter interfaced to a computer for data acquisition.

DURATION
- Preincubation period: Aliquots of 1 x 10E5 viable hepatocytes were allowed to attach for approximately 2 h in WME containing 10% calf serum.
- Exposure: Cultures were rinsed and exposed in serum-free medium containing test compound and 10 µC/mL of H-thymidine
- Expression time (cells in growth medium): 18 to 20 h after exposure, the cultures were washed three times with phosphate buffered saline
- Fixation time (start of exposure up to fixation or harvest of cells): Cells on coverslips were swelled in sodium citrate and fixed in ethanol:glacial acetic acid (3:1). The fixed cultures were washed twice, air dried and mounted cell surface up on glass slides with permount. Slides were dipped in NTB-2 photographic emulsion in the dark, allowed to dry overnight and stored at 4°C in light-proof slide boxes containing desiccant for one week.

STAIN: After 7 d exposure time, autoradiographs were developed, washed, immersed in Fixer (Eastman Kodak), washed, dried, stained in Harris Alum hematoxylin followed by a dip rinse in acid alcohol, rinsing in running tap water and a dip rinse in ammonium water. The slides were then rinsed in running tap water, dipped in 70% ethyl alcohol, followed by a dip in eosin solution. The slides were then rinsed with 95% ethyl alcohol, followed by rinsing in 100% ethyl alcohol. The slides were air dried, and coverslipped in permount. Excess emulsion was scraped off.

NUMBER OF REPLICATIONS: Triplicate cultures were assessed.

NUMBER OF CELLS EVALUATED: 150 hepatocytes per concentration; 50 cells/coverslip

DETERMINATION OF CYTOTOXICITY
Yes; by low overall grain incorporation.
Evaluation criteria:
A correction coefficient was calculated by the following method: An area/grain ratio was obtained by visually scoring an area of each slide containing 3-5 nuclear grains and then obtaining an object area count with a Colony Counter. The total number of the visual count was divided by the total number of the object area counts. This value serves as a correction coefficient. The cytoplasmic grain count was quantitated by randomly selecting the highest of three nuclear-sized areas adjacent to each nucleus. This value was subtracted from the uncorrected nuclear grain count to determine the net nuclear grain (NNG) count. Replicative or schedule DNA synthesis was evidenced by nuclei blackened with grains too numerous to count. The data of the HPC/DNA Repair Assay are reported as mean grains/nucleus from the triplicate wells.
Solvent and/or untreated controls should have a NNG count <0 with 0-10% hepatocytes in repair. Also, the positive control, 2AAF, should yield a mean NNG count that is within one standard deviation of the mean historical value (See below) with 70-100% of the hepatocytes in repair.
Species / strain:
hepatocytes: isolated from a Fischer F344 rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The top concentration is based on a pre-test where toxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION: There was an increased number of autoradiograph grains in all hepatocytes at all dose levels evaluated including the untreated negative and positive controls. This increase number of grains has resulted in a higher mean ± S.D. for the negative and positive controls as well as for the dose levels evaluated.

Table 1: Autoradiographic Analysis of DNA Repair in the Rat Hepatocyte Primary Culture

Treatment

NNG ± SD/150 Hepatocytes

Percent of Cells in Repair**

Untreated

-18.8 ± 10.5

0.7

2AAF (100 nM)

26.3 ± 17.5*

97.3

DMSO (1%)

-14.9 ± 13.3

7.3

0.25 (µg/mL)

-18.9 ± 13.9

8.0

0.5 (µg/mL)

-22.0 ± 17.5

6.0

1.0 (µg/mL)

-11.4 ± 13.2

14.7

2.5 (µg/mL)

-17.9 ± 12.3

4.0

*Positive finding. Mean NNG count >5 than the vehicle control.

**The percentage of cells that have NNG count >5.

Conclusions:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Not all results are displayed.
GLP compliance:
not specified
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
n.a.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
5, 15, 50 µg/mL
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin, 5 µg/mL (-S9); cyclophosphamide, 50µg/mL (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Evaluation criteria:
As described in guideline.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
negative

Executive summary:

No enhanced aberration rate in the S9 treated or untreated cells was observed in this chromosomal aberration assay. Therefore, the test substance was not considered to be clastogenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
0, 0.25, 0.75, 1.0, 1.5, 2.0 mg/mL (-S9)
0, 0.1, 0.25, 0.75, 1.0, 1.5, 2.0, 2.5 mg/mL (+S9)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate; dimethylnitrosamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Evaluation criteria:
As described in guideline.
Statistics:
yes
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation assay
Specific details on test material used for the study:
Trade Name: Marlipal 1618/1
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
dose finding test: 0, 1 - 100 µg/mL (solubility limit)
main test: 0, 1.8, 6, 18, 60, 100 µg/mL
Vehicle / solvent:
H0 medium with 1% ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H0 medium with 1% ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate (300 µg/mL), 3-methylcholanthrene (10 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Evaluation criteria:
According to Guideline.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
GLP compliance:
not specified
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Source, key, 9002-92-0, 1990
Conclusions:
interpretation of results: negative
Executive summary:

The potential of chromosome aberrations in mammalian cells of the target substance is estimated based on an adequate and reliable in vitro study of a structural analogue source substance. Experiments have been performed both in the presence as well as in the absence of metabolic activation in chinese hamster ovary (CHO) cells. All results obtained are negative, i.e. no chromosome aberrations in the cells investigated were observed. Therefore, no hazard with regard to chromosome aberrations in mammalian cells is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential to form chromosome aberrations.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Species / strain:
hepatocytes: isolated from a Fischer F344 rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The top concentration is based on a pre-test where toxicity was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source, key, 68439-50-9 (3EO), 1991b
Conclusions:
interpretation of results: negative
Executive summary:

The potential to induce DNA damage and/or repair in vitro of the target substance is estimated based on an adequate and reliable in vitro study of a structural analogue source substance. Experiments have been performed using hepatocytes isolated from a Fischer F344 rat. All results obtained are negative, i.e. no indication of unscheduled DNA synthesis was detected. Therefore, no hazard with regard to DNA damage is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential of gene mutations in mammalian cells.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Species / strain:
other: S. Thyphimurium TA 1535, TA1537, TA1538, TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative
Executive summary:

The potential of genetic toxicity in bacteria of the target substance is estimated based on adequate and reliable in vitro studies of structural analogue source substances. Experiments have been performed in Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 using the Ames plate incorporation method in triplicates, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

All results obtained are negative, i.e. no dose related increase in the number of revertantsg in the bacteria investigated were observed. Therefore, no hazard with regard to genetic toxicity to bacteria is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential of genetic toxiicty to bacteria.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Source, WoE, 68439-49-6, 1995b
Conclusions:
interpretation of results: negative
Executive summary:

The potential of gene mutation in mammalian cells of the target substance is estimated based on adequate and reliable in vitro studies of structural analogue source substances. Experiments have been performed both in the presence as well as in the absence of metabolic activation in chinese hamster ovary cells. Mutations in the HPRT locus were investigated. All results obtained are negative, i.e. no gene mutations in the cells investigated were observed. Therefore, no hazard with regard to gene mutation in mammalian cells is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the potential of gene mutations in mammalian cells.

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

Genetic toxicity in vivo

Description of key information

In vivo Mammalian Bone Marrow Chromosome Aberration Test (OECD guideline 475): negative

In vivo Mammalian Erythrocyte Micronucleus Test (OECD guideline 474): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to Guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
other: chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
According to Guideline.
Route of administration:
oral: gavage
Vehicle:
corn oil
Duration of treatment / exposure:
n.a.
Frequency of treatment:
single
Post exposure period:
12, 24, 48 h
Dose / conc.:
375 mg/kg bw/day (nominal)
Remarks:
(males)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
(males)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
(males)
Dose / conc.:
450 mg/kg bw/day (nominal)
Remarks:
(females)
Dose / conc.:
900 mg/kg bw/day (nominal)
Remarks:
(females)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
(females)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (30 mg/kg bw)
Tissues and cell types examined:
Femoral bone marrow smears
Details of tissue and slide preparation:
Sampling time: 12, 24, 48 h


Evaluation criteria:
According to Guideline.
Statistics:
Yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 410-1000 mg/kg bw
Conclusions:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals or test system and environmental conditions:
According to Guideline.
Route of administration:
intraperitoneal
Vehicle:
corn oil
Duration of treatment / exposure:
n.a.
Frequency of treatment:
single
Post exposure period:
30, 48, 72 h
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
640 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylenemelamine (0.3 mg/kg)
Tissues and cell types examined:
Tail blood smears
Details of tissue and slide preparation:
Sampling time: 30, 48, 72 h
Evaluation criteria:
According to Guideline.
Statistics:
Yes
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 410-1000 mg/kg bw
Conclusions:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Massachusetts, USA
- Age at study initiation: Pre-test: 9.5 weeks, Main study: 7 weeks
- Weight at study initiation: Pre-test: 37-43 and 30-33 g for males and females, respectively; Main study: 31-34 and 25-29 g for males and females, respectively
- Assigned to test groups randomly: yes, under following basis: body weight
- Housing: 5 per cage
- Diet (ad libitum): Wayne Lab Rodent Blox
- Water (ad libitum): Tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 3
- Humidity (%): 30 to 70
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: corn oil
- Lot/batch no. : Lot# 19F-003 (Sigma Chemical Company)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed and diluted with corn oil just prior to dosing.
Intraperitoneal administration was chosen to maximize bioavailability of the test item to the hematopoietic cells of the bone marrow.
Duration of treatment / exposure:
Single application
Frequency of treatment:
Single application
Post exposure period:
Mice were sacrificed 24, 48 and 72h after application of the test item.
Mice of the vehicle control were sacrificed 48 h after application.
Mice of the positive control were sacrificed 24 h after application.
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Pre-test: 2
Main study: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
triethylenemelamine (TEM) in 0.9% saline
- Dose: 0.5 mg/kg bw
Tissues and cell types examined:
Tissue: bone marrow
Cells: polychromatic erythrocytes and normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a preliminary Dose-Range-Finding study, the test item was administered intraperitoneally to 5 groups of CD-1 mice at 50, 100, 250, 500 and 1000 mg/kg bw. Due to the mortality observed at the two highest doses evaluated and the severe pharmacotoxic signs observed at the dose of 250 mg/kg bw, 100 mg/kg bw was selected as the dose in the MNT, as an estimate of the maximum tolerated dose.

DETAILS OF SLIDE PREPARATION:
Bone marrow of the femur was collected in fetal bovine serum and centrifuged. The pelleted bone marrow cells were resuspended in FBS and smear slides were preapred. Slides were dried, dipped in absolute methanol and air dried. Dried slides were stained using polychrome methylene blue and eosin, and a staining machine. Slides were coverd by permaslip and randomly coded.
Evaluation criteria:
Criteria for Scoring Micronuclei:
Micronuclei are uniform, darkly stained, typically round bodies in the cytoplasm of erythrocytes. Occasionally, micronuclei appear almond or tear drop shaped, inclusions in PCEs which are reflective, improperly shaped or stained, or which are not in the focal plane of the cell were judged to be artifacts and were not scored. Cells containing more than one micronucleus are scored as one micronucleated erythrocyte.
Criteria for a Valid Test:
If the spontaneous rate of micronuclei in the PCE is less than 0.5% and the positivec ontrol is statistically significant higher( p<0.05) than the spontaneous rate and at least seven animals per group survived the treatment, the results are deemed acceptable.
Statistics:
One-tailed t-tests were used to make pairwise comparisons between each treatment group and the vehicle control for statistically significant increases in the number of micronucleated PCE. The ratio of PCE/NCE was calculated based on 1000 erythrocytes for eacha nimal.The proportion of PCE per 1000 erythrocytesper animal was evaluated by pairwise two-tailed t-tests after an arcsin-transformation was performed.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs of toxicity were observed. However, the PCE/NCE ratio was not decreased but increased after 48 h treatment.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Immediately after dosing, 9/15 males and 8/15 females had decreased body tone and 6/15 males had piloerection. At 24 h, 5/15 males and 6/15 females had decreased body tone and 8/15 males and 4/15 females had piloerection. Body drop was seen in 1/15 males and 1/15 males had abnormal gait. At 48 h, 3/10 males and 2/10 females had decreased body tone and 3/10 males and 1/10 females had piloerection. At 72 h, 1/5 males had body drop, decreased body tone and piloerection.
No pharmacotoxic signs were observed in animals administered the positive or vehicle controls.
The vehicle and the positive control were within the historical control range.

Table 3: Results of the in vivo micronucleus assay

Treatment group

Dose

[mg/kg bw]

Sampling time

[h]

Mean frequency of PCE with MN

 

PCE/NCE ratio

Vehicle control

0

48

0.04

1.24

Test substance

100

24

0.05

0.98

48

0.03

1.64 (p≤0.05)

72

0.05

1.45

Positive control (TEM)

0.5

24

2.04 (p≤0.01)

0.84 (p≤0.05)

Conclusions:
negative
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Source, key, 112-59-4, 2001
Conclusions:
interpretation of results: negative
Executive summary:

The potential of the target substance for in vivo chromosome aberration in bone marrow is estimated based on an adequate and reliable in vitro key study of a structural analogue source substance. A single exposure of male and female Sprague-Dawley rats by oral gavage to doses up to 1500 mg/kg bw did not result in genotoxic effects in femoral bone marrow cells. Therefore, no hazard with regard to in vivo clastogenic effects is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the genotoxic potential.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
Summary of available data used for the endpoint assessment of the target substance
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to the category justification provided in IUCLID Section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
No. of animals per sex per dose:
5
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Source, WoE, 112-59-4, 2001
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical signs of toxicity were observed. However, the PCE/NCE ratio was not decreased but increased after 48 h treatment.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Source, WoE, 68439-50-9 (3EO), 1990c
Conclusions:
interpretation of results: negative
Executive summary:

The potential to induce the formation of erythrocyte micronuclei in vivo of the target substance is estimated based on two adequate and reliable in vivo studies of structural analogue source substances. Intraperitoneal administration of doses up to 640 mg/kg bw did not induce erythrocyte micronuclei in male and female mice. Therefore, no hazard with regard to in vivo erythrocyte micronuclei formation is identified for the target substance. As explained in the category justification, the differences in molecular structure between the target and the source substances are unlikely to lead to differences in the genotoxic potential.

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

Additional information

To cover the endpoint genotoxicity of the substance Decan-1-ol, ethoxylated (CAS 2618352 -8), studies from similar substances were taken for read-across. Read-across is justified because the length of the alkyl chain and the grade of ethoxylation does not exert any meaningful influence on genotoxicity. Moreover, the structure of alcohol ethoxylates (AE) is not of concern for potential genotoxicity and in all available in-vitro and in-vivo genotoxicity assays, there was no indication of genetic toxicity of broad range of structurally different AE (HERA, 2009). Studies which were undertaken in vitro using a number of test systems have resulted in negative findings.

 

Genetic toxicity in vitro and in vivo

The results of several tests with AE using several strains of S. typhimurium or E. coli, were all negative. Testing with mammalian cell lines for a chromosomal aberration assay with AE C16-18 gave a negative result as did the mouse lymphoma assay with AE C16-18 and AE C6-10. The result of in-vivo testing in murine micronucleus assay and a chromosal aberration test with AE C6-10 were as well negative. Based on the outcome of these tests, AE can be considered as non-genotoxic.

Justification for classification or non-classification

According to the classification criteria of Regulation (EC) No. 1272/2008 the substance does not need to be classified for genetic toxicity.