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EC number: 826-704-5 | CAS number: 1631962-93-0
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
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- Density
- Particle size distribution (Granulometry)
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- Oxidation reduction potential
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- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
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- Carcinogenicity
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- Exposure related observations in humans
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- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- This study was conducted between 16 December 2014 and 18 February 2015
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Well conducted and well described study in accordance with GLP and OECD guideline 473 without any deviation
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- 2014
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- 1-(2-methylpropyl)-4-(propoxymethyl)benzene
- EC Number:
- 826-704-5
- Cas Number:
- 1631962-93-0
- Molecular formula:
- C14H22O
- IUPAC Name:
- 1-(2-methylpropyl)-4-(propoxymethyl)benzene
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- Identity: FRET 11-0539
Chemical name: 1-(2-methylpropyl)-4-(propoxymethyl)-benzene
Molecular weight: 206.17
Batch number: RB272-88
Expiry: December 2015
Appearance: Clear colourless liquid
Storage conditions: Room temperature, dark
Purity: >99%
Correction factor for formulation: None, used as supplied
Date received: 21 November 2014
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: cultures prepared from the pooled blood of two healthy, non-smoking, adult donors
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C.
- Test concentrations with justification for top dose:
- The final concentrations to which cells were exposed initially are given below. Those cultures that were analysed for chromosome aberrations areindicated *.
Preliminary toxicity test: 12.09, 20.16, 33.59, 55.99, 93.31, 155.52, 259.2, 432, 720 and 1200 g/mL
Main tests: -S9 mix (3 hours) 20*, 25, 30*, 35, 40*, 45 and 50 µg/mL
+S9 mix (3 hours) 90, 100, 110, 120, 130, 140 and 150 µg/mL
-S9 mix (21 hours) 5*, 10, 15, 20*, 22.5*, 25, 27.5 and 30 µg/mL
Additional Main test: +S9 mix (3 hours) 100, 110, 120, 130, 140*, 150, 160*, 170, 180, 190* and 200 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Details on test system and experimental conditions:
- PREPARATION OF CULTURES: Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with HML media. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen. Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), HML media (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37°C, and the cells resuspended (twice daily) by gentle inversion.
S9 metabolizing system
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C. The quality control statements relating to the batches of S9 preparation used are included in the raw data and copies are included in this report as Annex 3.
Lot No.: 3318 (Date of preparation: 31 July 2014) – preliminary toxicity test
Lot No.: 3350 (Date of preparation: 8 October 2014) – main test
Preparation of S9 mix
S9 mix contained: S9 fraction (25% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter sterilised before use.
Preliminary toxicity test procedure
Cultures were treated approximately 48 hours after commencement of incubation of lymphocyte cultures. All cultures were uniquely identified. Cultures were prepared for each treatment (3-hour treatment in the absence and presence of S9 mix, and 21-hour continuous treatment in the absence of S9 mix).
Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with the test substance for each test condition. No positive control cultures were prepared.
All cultures were centrifuged and resuspended in fresh medium before treatment, and S9 homogenate was present in appropriate cultures at a final concentration of 5% v/v.
FRET 11-0539 was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control.
At the end of the 3-hour treatment period, cultures were examined for the presence of precipitate. Cultures were then centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then washed in saline (5 mL), resuspended in fresh medium, and incubated (for approximately 18 hours) until the scheduled harvest time.
At the end of the 21-hour treatment period, cultures were examined for the presence of precipitate.
Harvesting and fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The cell pellets were treated with a hypotonic solution (0.075M KCl), pre-warmed at 37°C. After a 10 minute period of incubation at 37°C, the suspensions were centrifuged at 500g for 5 minutes and the cell pellets fixed by addition of freshly prepared cold fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced until it was clear.
Slide preparation
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. One slide was prepared per culture. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.
Microscopic examination
The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed. Slides were assessed for a depression in mitotic index (except when clear evidence of overt toxicity is observed, or in cultures where there were no signs of cytotoxicity).
Main test procedure
The procedure for the main tests was the same as that for the preliminary tests, with the following exceptions: positive control cultures were included for all tests; duplicate cultures were prepared for all cultures. Two slides were prepared per culture.
3-hour treatment in the absence of S9 mix
FRET 11-0539 was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C was the positive control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium (final volume of 5 mL). They were then incubated for a further 18 hours. The cultures were then harvested and slides prepared.
3-hour treatment in the presence of S9 mix
FRET 11-0539 was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Cyclophosphamide was the positive control.
Following 3-hour treatment, cultures were centrifuged at 500g for 5 minutes and the supernatant removed. Cultures were then resuspended in saline and centrifuged at 500g for 5 minutes. The saline was then removed and the cell pellets resuspended in fresh medium (final volume of 5 mL). They were then incubated for a further 18 hours. The cultures were then harvested and slides prepared.
After 3-hour treatment in the presence of S9 mix, the required toxicity to permit metaphase analysis was not achieved. An additional test was conducted using a modified concentration range.
21-hour treatment in the absence of S9 mix
FRET 11-0539 was added to each culture in 50 µL aliquots. Ethanol was used as the vehicle control, and Mitomycin C was the positive control.
Following the end of the treatment period the cultures were harvested and slides prepared.
Microscopic examination
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded (except for when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).
From these results the concentration causing a decrease in mitotic index of 45% 5% when compared to the vehicle control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.
The selected slides were then coded. Metaphase cells were identified using a low power objective and examined at a magnification of x1000 using an oil immersion objective. One hundred and fifty metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 cells in 150 metaphases with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. The vernier readings of all aberrant metaphase figures were recorded.
The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded as a percentage of the 150 metaphases analysed per slide.
The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).
A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.
D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). The following model was used:
p = C + (1 – C) / 1 + exp{– intercept – slope ln(conc)}
p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.
The D20 values for structural aberrations (including and excluding gaps) are retained within the study data.
The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002). - Evaluation criteria:
- An assay was considered to be acceptable if the negative and positive control values lie within the current historical control range.
The test substance was considered to cause a positive response if the following conditions were met:
Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) were observed at one or more test concentration.
The increases exceed the vehicle control range of this laboratory, taken at the 95% confidence limit.
The increases were reproducible between replicate cultures.
The increases were not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
Evidence of a concentration-related response was considered to support the conclusion.
A negative response was claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.
A further evaluation would be carried out if the above criteria for a positive or a negative response are not met.
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test
Toxicity data
In the absence of S9 mix following 3-hour treatment, FRET 11-0539 caused a reduction in the mitotic index to 14% of the vehicle control value at 55.99 µg/mL. At higher tested concentrations overt toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.
In the presence of S9 mix following 3-hour treatment, FRET 11-0539 caused a reduction in the mitotic index to 42% of the vehicle control value at 155.52 µg/mL. At higher tested concentrations overt toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.
In the absence of S9 mix following 21-hour continuous treatment, FRET 11-0539 caused a reduction in the mitotic index to 24% of the vehicle control value at 33.59 µg/mL. At higher tested concentrations overt toxicity was observed. No notable culture medium changes were observed, when compared to the vehicle control.
The concentrations used in the main test were based upon these data.
Main test: 3-hour treatment in the absence of S9 mix
Toxicity data
No notable culture medium changes were observed, when compared to the vehicle control.
FRET 11-0539 caused a reduction in the mitotic index to 49% of the vehicle control value at 40 µg/mL. The concentrations selected for metaphase analysis were 20, 30 and 40 µg/mL.
Metaphase analysis
FRET 11-0539 caused a statistically significant increase (p<0.05) in the proportion of metaphase figures containing chromosomal aberrations at 30 µg /mL (including gaps only) and at 40 µg /mL (excluding gaps only). The increases were not dose-related and were within the laboratories historical vehicle control values. There were no other statistically significant increases observed. The increases are therefore considered not to be biologically relevant.
All mean values for the vehicle control (ethanol), and all FRET 11-0539 treatment concentrations were within the laboratory historical control range, when taken at the 95% confidence limit.
The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.
Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.
Main test: 3-hour treatment in the presence of S9 mix
Toxicity data
No notable culture medium changes were observed, when compared to the vehicle control.
FRET 11-0539 caused a reduction in the mitotic index to 84% of the vehicle control value at 150 µg/mL. As the required toxicity range to permit metaphase analyses was not achieved, an additional test was conducted using a modified concentration range.
Additional Main test: 3-hour treatment in the presence of S9 mix
Toxicity data
No notable culture medium changes were observed, when compared to the vehicle control.
FRET 11-0539 caused a reduction in the mitotic index to 43% of the vehicle control value at 190 µg/mL. The concentrations selected for metaphase analysis were 140, 160 and 190 µg/mL.
Metaphase analysis
FRET 11-0539 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle control.
All mean values for the vehicle control (ethanol), and all FRET 11-0539 treatment concentrations were within laboratory historical control range, when taken at the 95% confidence limit.
The positive control compound, Cyclophosphamide, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.
Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.
Main test: 21-hour continuous treatment in the absence of S9 mix
As FRET 11-0539 was negative for both short term treatments (3-hour treatment in the absence and presence of S9 mix), in accordance with current guidelines, cultures treated continuously for 21 hours in the absence of S9 mix underwent metaphase analysis.
Toxicity data
No notable culture medium changes were observed, when compared to the vehicle control.
FRET 11-0539 caused a reduction in the mitotic index to 48% of the vehicle control value at 22.5 µg/mL. The concentrations selected for metaphase analysis were 5, 20 and 22.5 µg/mL.
Metaphase analysis
FRET 11-0539 caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle control.
All mean values for the vehicle control (ethanol), and all FRET 11-0539 treatment concentrations were within the laboratory historical control range, when taken at the 95% confidence limit.
The positive control compound, Mitomycin C, caused statistically significant increases (p<0.001) in the proportion of aberrant cells. This demonstrated the sensitivity of the test system.
Polyploidy and endoreduplication analysis
No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control. - Remarks on result:
- other: all strains/cell types tested
Any other information on results incl. tables
Table1 Summary of results
Exposure period |
S9 mix |
Nominal concentration of FRET 11-0539 |
Cells with aberrations excluding gaps |
Cells with aberrations including gaps |
Relative Mitotic |
||||
(hours) |
|
(µg/mL) |
Individual values (%) |
Mean (%) |
Individual values (%) |
Mean (%) |
Index (%) |
||
3 |
- |
0 (Ethanol) |
1.3 |
0.0 |
0.7 |
1.3 |
2.0 |
1.7 |
100 |
|
|
20 |
0.7 |
0.7 |
0.7 |
0.7 |
2.7 |
1.7 |
107 |
|
|
30 |
0.7 |
3.3 |
2.0 |
2.7 |
5.3 |
4.0* |
73 |
|
|
40 |
1.3 |
4.0 |
2.7* |
1.3 |
6.0 |
3.7 |
49 |
|
|
0.2 (Mitomycin C) |
21.1 |
31.3 |
25.2*** |
23.9 |
35.4 |
28.6*** |
103 |
|
|
|
|
|
|
|
|
|
|
3 |
+ |
0 (Ethanol) |
0.7 |
1.3 |
1.0 |
1.3 |
1.3 |
1.3 |
100 |
|
|
140 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
105 |
|
|
160 |
0.7 |
0.0 |
0.3 |
1.3 |
2.0 |
1.7 |
71 |
|
|
190 |
0.7 |
1.3 |
1.0 |
2.0 |
3.3 |
2.7 |
43 |
|
|
7.5 (Cyclophosphamide) |
26.8 |
25.9 |
26.3*** |
32.1 |
25.9 |
28.9*** |
44 |
|
|
|
|
|
|
|
|
|
|
21 |
- |
0 (Ethanol) |
2.7 |
0.0 |
1.3 |
4.0 |
2.0 |
3.0 |
100 |
|
|
5 |
0.0 |
0.0 |
0.0 |
1.3 |
1.3 |
1.3 |
115 |
|
|
20 |
0.7 |
0.7 |
0.7 |
3.3 |
2.0 |
2.7 |
68 |
|
|
22.5 |
3.3 |
1.3 |
2.3 |
4.7 |
3.3 |
4.0 |
48 |
|
|
0.1 (Mitomycin C) |
34.1 |
32.6 |
33.3*** |
34.1 |
32.6 |
33.3*** |
105 |
One-tailed Fisher's exact test
*** p<0.001
* p<0.05
Otherwise p>0.05
Applicant's summary and conclusion
- Conclusions:
- It is concluded that the test substance FRET 11-0539 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
- Executive summary:
A study was performed to assess the ability of FRET 11-0539 to cause structural chromosome aberrations in human lymphocytes cultured in vitro.
Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.
A preliminary toxicity test was performed. A 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix were used to determine toxicity. Based on the mitotic index data, concentrations were selected for the main test.
In the main test, the mitotic index was assessed for all cultures treated with FRET 11-0539 and the vehicle control, ethanol. Justification for the highest analysed concentration was determined by cytotoxicity.
On the basis of these data, the following concentrations were selected for metaphase analysis:
In the absence of S9 mix, 3-hour treatment: 20, 30 and 40 µg/mL
In the presence of S9 mix, 3-hour treatment: 140, 160 and 190 µg/mL
In the absence of S9 mix, 21-hour continuous treatment: 5, 20 and 22.5 µg/mL
In the absence of S9 mix following 3-hour treatment, FRET 11-0539 caused a statistically significant increase (p<0.05) in the proportion of metaphase figures containing chromosomal aberrations at 30 microgram/mL (including gaps only) and at 40 microgram/mL (excluding gaps only). The increases were not dose-related and within the laboratories historical vehicle control values. There were no other statistically significant increases. The increases are therefore considered not to be biologically relevant.
In the presence of S9 mix following 3-hour continuous treatment, FRET 11-0539 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.
In the absence of S9 mix following 21-hour continuous treatment, FRET 11-0539 caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.
No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.
Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.
It is concluded that FRET 11-0539 has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.
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