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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames Assay: Negative with and without metabolic activation S. typhimurium strains TA98, TA100, TA1535 and TA1537, and E.coli strain WP2uvrA.

Chromosome Aberration Test: Ambigous results in CHL with and without metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 April 1989 to 18 May 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in compliance with GLP.
Qualifier:
according to guideline
Guideline:
other: "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour,
Principles of method if other than guideline:
The study was conducted in compliance with "Good Laboratory Practice (GLP) Standards as set forth in Article 4 relating to Test Items of New Chemical Substances and Survey Items of Toxicity of Designated Chemical Substances'' issued by the Japanese Ministry of International Trade and Industry (MITI) etc. and "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour, and the report presents a full and accurate account of the results of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidene
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:
E. coli WP2 uvr A
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 determination test: 1, 5, 10, 50, 100, 500, 1000, 5000 μg/plate
Main test: 156, 313, 625, 1250, 2500, 5000 μg/plate
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2- (2- Fury I)-3- (5-nitro-2-furyl)- acrylamide (AF- 2); 2- Aminoanthracene ( 2- AA)
Details on test system and experimental conditions:
Test substance: 2,4,8,10-Tetrakis (1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d.g] [1,3,2] dioxaphosphocin [other name: 2,2'-methylenebis(4,6-di-tertbutylphenyl)2-ethylhexyl phosphite] was suspended in dimethylsulfoxide (DMSO) by uitrasonification.
Bacterial strains: Salmonella typhimurium stranis TA98, TA100. TA1535 and TA1537 and Escherichia coli WP2uvrA were supplied by Dr. T. Matsushima, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo.
Bacterial cultures in Oxoid nutrient broth #2 were freshly prepared before use by inoculating bacteria from frozen stock cultures (kept at -80°C).
Fifty μl stock solution was inoculated into the 15ml broth and incubated for 10 hours at 37°C and 180rpm using rotary shaker.
S9 and S9Mix;: S9, post-mitochondrial supernatant of rat-liver homogenates was obtained from Kikkoman Corporation. Chiba (JAPAN). This S9 was prepared by slight modifications of method of Ames [1] :5.6-Benzoflavone and Phenobarbital were used as inducers of drug-metabolizing enzyme system [2]. The S9mix contained 4mM NADPH, 4mM NADH. SmM G-6-P. 8mM MgCl2, 33mM KCI. 100mM sodium phosphate buffer (pH 7.4) and 10% S9 (50 μl S9 per plate).
Evaluation criteria:
Not specified in the study report.
Statistics:
Not specified in the study report.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1 ,3,2] dioxaphosphocin did not induce revertant colonies more than twice of that of the solvent control on the S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2uvrA with or without metabolic activation.
Positive control substances showed increases in revertant colony count more than twice of the solvent control values with bacterial strains in various tests, and these suggested that susceptibilities of test bacterial strains were adequately maintained and the test was adequately carried out.
Based on the above results, the present test substance was assessed to be negative.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Based on the results, the present test substance was assessed to be negative.
Executive summary:

Purpose of this test is an evaluation of the mutagenicity of 2,4,8,10-tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo [d.g] [1,3,2]dioxaphosphocin [other name: 2,2'-Methylenebis(4,6-di-tert-butylphenyl)2-ethylhexyl phosphite] by the microbial mutagenicity test.

 

The study was conducted in compliance with "Good Laboratory Practice (GLP) Standards as set forth in Article 4 relating to Test Items of New Chemical Substances and Survey Items of Toxicity of Designated Chemical Substances'' issued by the Japanese Ministry of International Trade and Industry (MITI) etc. and "Standards to be observed by Mutagenicity Testing Institutions" issued by the Japanese Ministry of Labour, and the report presents a full and accurate account of the results of the study.

2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1,3,2] dioxaphosphocin did not induce revertant colonies morethan twice of that of the solvent control on the S. typhimurium TA98,TA100, TA1535 and TA1537, and E. coli WP2uvrA with or without metabolic activation.

Positive control substances showed increases in revertant colony countmore than twice of the solvent control values with bacterial strains in various tests, and these suggested that susceptibilities of test bacterial strains were adequately maintained and the test was adequately carried out.

Based on the above results, the present test substance was assessed to be negative.

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:
22 September 1989 to 8 December 1989
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
other: "Guideline of Chromosomal Aberration Test in Cultured Mammalian Cells" by means of the "Guidelines for Screening Toxicity Testings of Chemicals", Environment Agency, Ministry of Health and Welfare, Ministry oflnternational Trade and Industry, Japan, 1986.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
polyploids
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
A clonal sub-line derived from the lung of a female newborn Chinese hamster (CHL) was kindly supplied by Dr.M.Ishidate, Jr., of the National Institute of Hygienic Science, Tokyo, Japan in December 14, 1985.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
The tests without metabolic activation (24 and 48 hours treatments) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml).
Tests with metabolic activation (6 hours treatment) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) in both -S9 and +S9 treatments.
Vehicle / solvent:
The test substance was soluble in tetrahydrofuran(THF), but the test substance formed large precipitate when the test substance solution in THF added into culture medium, consequently THF was not used for solvent in this study. The test substance was hardly soluble in other solvents (DMSO, ethanol, acetone etc. ), so they were not used for solvent. The reason of above-mention, the test substance was suspended in culture medium directly in this study.

Culture condition
Medium: Eagle's MEM (GIBCO Laboratories) supplemented with 10% heat-inactivated calf serum (CS, Hyclone, Lot No. 2151719).
Culture: 5% C02, 37°C, subcultured by 2-4 day, growth in monolayer.
Doubling time is approximately 15 hours.
The modal chromosome number is 25.
Storage: The cells that were suspended in medium with 10 % DMSO were poured into vials for freezing and were stored at –197 °C (Liquid nitrogen).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
Preparation of the test substance: The test substance was ground into fine powder by a mortar, and the desired amount of the test substance was weighted and the highest dosing suspension was prepared by adding the appropriate volume of culture medium, and sonication was performed for dispersion. The other doses were prepared by performing serial dilution by culture medium. Treatment was performed by change culture medium to the test substance suspension medium.
The handling of the test substance was conducted under a safety lamp (yellow coloured lamp).

Metabolic activation system
Suppliers of S9:
Source: Purchase (Manufacturer: Kikkoman Co., Ltd.)
Prepared on: June 23, 1989
Purchased on: August 2, 1989
Lot No.: RAA – 223
Storage temperature: -80°C
Amount of protein: 27.7 mg/ml
S9 was used for the test within 6 months after preparation.

Preparation of S9
Animal used
Species: Rat
Strain: SD
Sex: Male
Age: 7 weeks
Bodyweight: 193 – 236 g

Inducing substance
Name: Phenobarbital (PB) and 5,6-Benzoflavone (BF)
Injection: Intraperitoneally
Amount and Administration period (g/kg – bodyweight): 1st (initiation day): PB 0.03 (g/kg BW); 2-4th: PB 0.06 9g/kg BW); 3rd: BF 0.08 (g/kg BW).
The rat was sacrificed 5 day after the initiation of Phenobarbital administration, and the liver was perfused with cold saline and homogenized with 0.15M KCl of three times volume of weight of the liver. The homogenates were centrifuged at 9000 G for 10 minutes and the supernatants were used as S94l. The S9 was then purchased for use in the test.

Composition of S9 mix
Constituents Amount in 1ml S9 mix
S9 0.3 ml
MgCl2 5 μ mol
KCI 33 μ mol
G-6-P 5 μ mol
NADP 4 μ mol
HEPES buffer 4 μ mol
Distilled Water -
The concentration of the S9 fraction in the final treatment medium was 5 %

Test method
Adopt a test method: The test was conducted according to the Guideline of "Chromosomal
Aberration Test in Cultured Mammalian Cells" by means of the "Guidelines for Screening Toxicity Testings of Chemicals", Environment Agency, Ministry of Health and Welfare, Ministry of lnternational Trade and Industry, Japan, 1986.

Cell growth inhibition test: The cell growth inhibition test, which was used for checking cytotoxicity, was carried out in order to determine the treatment levels of the test substance in the chromosomal aberration test. In this test two culture dishes for each concentration level were used for with and without metabolic activation method.
Without metabolic activation(24 and 48 hours treatments): 2E4 Cells were seeded in a 60 mm plastic Petri dish and cultured for three days, then the culture medium was changed to the test substance suspension medium. Cells treated with culture medium alone were served as negative control. After 24 or 48 hours treatments, the medium was discarded and the cells were washed with physiological saline. Ethanol was added to fix the cells which were then stained with 0.1 % crystal violet. After washing and drying each dishes were placed under a cell densitometer (Monocellater, Olympus Co., Ltd.) to measure the coloUr absorption value (555 nm). The cell growth index was calculated with the control being 100 % and no live cell being 0 %.
With metabolic activation (6 hours treatment): For metabolic activation, the 89 mix was added to the culture medium for 6 hours treatment.
2E4 Cells were seeded in a 60 mm plastic Petri dish, and cultured for three days, then the culture medium was changed to the test substance suspension medium with 89 mix, and cultured for 6 hours. Then, test substance suspension medium with S9 mix was changed to fresh culture medium. Cells treated with culture medium alone were served as negative control. Fixation and staining were made after additional culture for 18 hours.

Preliminary chromosomal aberration test (Test for determining concentration levels): Preliminary chromosomal aberration test was performed at the selected concentration levels on the basis of the cell growth inhibition test. One culture dish was used for each dose levels in preliminary chromosomal aberration test.
Without metabolic activation (24 and 48 hours treatments): 2E4 Cells were seeded in a 60 mm plastic Petri dish and cultured for three days. The culture medium was changed to the test substance suspension medium. Test substance was allowed to remain in the cultures until chromosome preparations were made at 24 or 48 hours after the start of the treatment. Cells treated with culture medium alone were served as negative control and MMC as positive control. For this study, six concentration levels of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml of the test substance were used.
With metabolic activation (6 hours treatment): For metabolic activation, the S9 mix (+S9) was added to the culture medium for 6 hours treatment. As a control, a test without addition of the S9 mix (-S9) was carried out at the same concentration levels.
2E4 Cells were seeded in a 60 mm plastic Petri dish, and cultured for three days. The culture medium was changed to the test substance suspension medium with S9 mix, and cultured for 6 hours. Then, test substance suspension medium with S9 mix was changed to fresh culture medium. Chromosome preparations were made after additional culture for 18 hours. Cells treated with culture medium alone were served as negative control and B[a]P as positive control. For this study, six concentration levels of 0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml of the test substance were used.

Chromosomal aberration test (main test): Main chromosomal aberration test was carried out at the selected concentration levels on the basis of the cell growth inhibition test and preliminary chromosomal aberration test. The procedure of chromosomal aberration test was the same way as described above.
In this main test, two culture dishes for each concentration level were used for with and without metabolic activation methods.
Slide preparation: Two hours before the end of culture, cells were treated with 0.211 g/ml Colcemid. And after finishing culture, they were dissociated with trypsin (0.05% trypsin + 0.02% EDTA) and centrifuged( 1000ppm, 5 min ) to collect cells. Then supernatant was removed, cells were incubated in 75 Mm hypotonic KCl for 20 minutes at 37 °C. The cells were then fixed three times with acetic acid-ethanol (1:3) and spread onto clean glass slides. Each slide was stained with Giemsa solution (2.5% at pH 6.8) for 12 minutes after air drying.

Chromosome observation and evaluation
Observation: The number of cells with chromosomal aberration from 100 metaphases were counted per each culture dish. The types of aberration were classified into 6 groups : chromatid and chromosome gaps (g), chromatid breaks (ctb), chromatid exchanges (cte), chromosome breaks (csb), chromosome exchanges (cse), and others (fragmentation, etc., excluding pulverization). The incidence of polyploid (pol) cells in the 100 metaphases was recorded.
Criteria for observation: The following criteria for observation were determined, based on the fact that the CHL cells are established cell and also taking into consideration the characteristics of the chromosomes.
-Gap: The achromatic region is located on the vertical axis of the chromatid, the width of which is either equal to or wider than the width of the chromatid. The shape of the achromatic region is clearly observed.
-Break: The broken section of the chromosome is not aligned with the vertical axis of the chromatid. Even if the section is located on the same axis, the achromatic region is twice as wide or wider than the width of the chromatid (in the case of thin and long chromosomes, however, this criteria is not applied.). With respect to the breakage of the chromosomal pattern, since there are chromosomes without the centromere, only clearly recognizable example is recorded.
-Exchange: By definition, this is two or more detached segments on one or more chromosomes, resulting from chromosome breakage, arranged in conjugated form.
As regards the chromosomal patterns, only clearly recognizable example, such as ring or dicentric chromosomes , is observed.
-Others:
Fragmentation: Chromosome examples in which many cases of gaps and chromosomal breaks are observed, excluding the exchange forms.
-Numerical aberration (polyploidy): Polyploidy: By definition, polyploidy examples include triploid (37 rods) or polyploidy examples greater than triploid. Aneuploidy is not investigated.
Evaluation criteria:
For the evaluation of the frequencies of structural aberrations and of polyploidy, the following criteria which are usually used for chromosomal aberration testing with CHL are employed.
Negative (-): less than 5%
Equivocal (±): from 5% to less than 10%
Positive (+): 10% or more,
The total frequencies of structural aberrations include the frequencies of aberrant cells which have only gaps.
Statistics:
Statistical analysis was not performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Cell growth inhibition test: The cell was treated with the test substance up to 5.0 mg/ml at the highest concentration. As the result, no strong cytotoxicity was observed at any concentration levels with and without metabolic activation.

Preliminary chromosomal aberration test: In this method, the metaphase cells were observed all the way up to the highest concentration level (5.0 mg/ml) in both 24 hours and 48 hours treatments. In the 24 hours treatment, no structural aberration was observed at any concentration levels. However, 5 and 5% polyploidy induction were observed at the concentration levels of 2.0 and 3.0 mg/ml, respectively. In the 48 hours treatment, no structural aberration was observed at any concentration levels. However, 8, 8 and 10% polyploidy induction were observed at the concentration levels of 3.0, 4.0 and 5.0 mg/ml, respectively. As a result, taking the test results of the cell growth inhibition also into account, the concentration levels of main chromosomal aberration test were determined at the five levels of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/ml in both the 24 hours and 48 hours treatments without metabolic activation.
In this method, the metaphase cells were observed all the way up to the highest concentration level (5.0 mg/ml) in both -S9 and +S9 treatments. Although in -S9 treatment, 5% of polyploidy induction was observed at the concentration levels of 1.0 and 3.0 mg/mL no structural aberration or polyploidy induction was found at the other concentration levels in both -S9 and +S9 treatments. Therefore, when taking the results of the cell growth inhibition test also into account, the concentration levels of main chromosomal aberration test were determined at the five levels of 1.0, 2.0, 3.0, 4.0, and 5.0 mg/ml.
Chromosomal aberration test (main test): In the 24 hours treatment, no structural aberration was observed at any concentration levels. However, 5.0, 4.5, 6.5 and 9.5 % polyploidy induction were observed at the concentration levels of 2.0, 3.0, 4.0 and 5.0 mg/ml, respectively. In the 48 hours treatment, no structural aberration was observed at any concentration levels. However, 10.5 and 8.5% polyploidy induction were observed at the concentration levels of 4.0 and 5.0 mg/ml, respectively.
In the -S9 treatment, no structural aberration was observed at any concentration levels. However, 7.0, 7.0, and 7.5 % polyploidy induction were observed at the concentration levels of 3.0, 4.0 and 5.0 mg/ml, respectively. Although in the + 89 treatment 5 % of polyploids was observed at the concentration level of 5.0 mg/ml, no structural aberration or polyploidy induction was found at other concentration levels.
The test substance was hardly soluble in the culture medium, and test substance was sedimented on the culture dish in the treatment concentration levels (0.5-5.0 mg/ml).
In all tests, the frequencies of chromosomal aberrations were very low in the negative control group whereas induction was clearly observed in the positive control groups. All the aberration frequencies were within the range of the historical data of this laboratory. The CHL used was normal, and the procedures including metabolic activation and treatment were carried out in the usual way. The validity of the test procedure can thus be assured.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
ambiguous without metabolic activation
negative with metabolic activation

It is concluded that the test substance has the property of inducing polyploidy in CHL cells in the 48 hours treatment without metabolic activation.
This conclusion can be derived from the fact that the rate of appearance of the polyploid cells when applying the 48 hours treatment is more than 10%, which is the criterion for positive evaluation.
Executive summary:

Chinese hamster lung cell line (CHL) was treated with 2,4,8,10-Tetrakis(1,1-dimethylethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1,3,2] dioxaphosphocine in order to investigate the ability of the test substance to induce chromosomal aberrations.

The test was conducted according to the Guideline of "Chromosomal Aberration Test in Cultured Mammalian Cells" by means of the "Guidelines for Screening Toxicity Testings of Chemicals", Environment Agency, Ministry of Health and Welfare, Ministry of lnternational Trade and Industry, Japan, 1986.

 

The tests without metabolic activation (24 and 48 hours treatments) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml).

As a result, in the 48 hours treatment, 10.5% and 8.5% polyploidy induction were observed at the concentration levels of 4.0 and 5.0 mg/ml, respectively. Further, in the 24 hours treatment, 5.0%, 4.5%, 6.5%, 9.5% polyploidy induction were observed at the concentration levels of 2.0, 3.0, 4,0 and 5.0 mg/ml, respectively.

No structural aberration was observed at any concentration levels in both 24h and 48h treatments.

Tests with metabolic activation (6 hours treatment) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) in both -S9 and +S9 treatments. As a result, in the -S9 treatment, 7.0%, 7.0 % and 7.5 % polyploidy induction were observed at the concentration levels of 3.0, 4.0 and 5.0 mg/ml, respectively. Further, in the +89 treatment, 5.0% polyploidy induction was observed at the concentration levels of 5.0 mg/ml. No structural aberration was observed at any concentration levels in both -S9 and + S9 treatment.

 

It is concluded that the test substance has the property of inducingpolyploidy in CHL cells in the 48 hours treatment without metabolic activation.

This conclusion can be derived from the fact that the rate of appearance of thepolyploid cells when applying the 48 hours treatment is more than 10%, which is the criterion for positive evaluation.

Genetic toxicity in vivo

Description of key information

Micronucleus Test: Negative in bone marrow cells of mice.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 August 1999 and 23 September 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD, EU and US EPA test guidelines in compliance with GLP.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
All animals in this study were Specific Pathogen Free CD-1 outbred albino mice of Swiss origin.
Males weighed between 28 and 30 grams and females weighed between 22 and 24 grams on despatch from Charles River UK Limited, Margate, Kent, England.
On arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and tail marked. Each group was kept, with the sexes separated, in disposable cages and maintained in a controlled environment, with the thermostat set at 22°C and relative humidity set at 50%. The room was illuminated by artificial light for 12 hours per day. All animals were allowed free access to pelleted expanded rat and mouse No.1 maintenance diet (SQC grade obtained from Special Diets Services Ltd, Witham, Essex, UK) and tap water ad libitum. Food and tap water are routinely analysed for quality at source. Dietary contaminants are not suspected of having any significant effect on parameters measured in this test in this laboratory at any time over the last ten years. All animals were acclimatised for a minimum of 5 days, examined daily and weighed prior to dosing.
Route of administration:
intraperitoneal
Vehicle:
Suspensions of the test substance were freshly prepared on the morning of the test (using identical methods for each phase of the test) and were individually formulated to the concentrations in corn oil.
Details on exposure:
All animals in all groups were dosed with the standard volume of 20 ml/kg bodyweight. The test substance and negative control were dosed by intraperitoneal injection, and mitomycin C, the positive control compound, was administered orally by intragastric gavage.
Duration of treatment / exposure:
24 & 48 hours exposure
Frequency of treatment:
Single treatment
Post exposure period:
up to 48 hours
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5 males & females for 500, 1000 & positive control.
10 males & 10 females for vehicle control & 2000 dose groups.
Control animals:
yes
Positive control(s):
Mitomycin C, obtained from Sigma Chemical Company, batch number 118H2500; was used as the positive control compound. It was prepared as a solution in purified water, at a concentration of 0.6 mg/ml, just prior to administration.
Tissues and cell types examined:
erythrocytes
Details of tissue and slide preparation:
The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum using a 2 ml disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). At least three smears were made from each animal. The prepared smears were fixed in methanol(> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1:9 dilution of Gurr's improved R66 Giemsa (BDH) with purified water).
Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with covers lips using DPX.
The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.

Micronuclei are identified by the following criteria:
• Large enough to discern morphological characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells - not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cell.
The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).
Evaluation criteria:
A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Bone marrow cell toxicity (or depression) is normally indicated by a substantial and statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time; a decrease at the 24 hour sampling time is not necessarily expected because of the relatively long transition time of erythroid cells [late normoblast -> immature erythrocyte (approximately 6 hours) -> mature erythrocyte (approximately 30 hours)].
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. Non-parametric statistical methods were chosen for analysis of results because:
• They are suited to analysis of data consisting of discrete/integer values with ties such as the incidence of micronucleated immature erythrocytes
• The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of nonparametric analysis and classical analysis of variance are very similar)
• 'Outliers' are frequently found in the proportion of immature erythrocytes for both control and treated animals; non-parametric analysis based on rank does not give these values an undue weighting.
Unless there is a substantial difference in response between sexes (which occurs only rarely) results for the two sexes are combined to facilitate interpretation and maximise the power, of statistical analysis.
For incidences of micronucleated immature erythrocytes, exact one-sided p-values are calculated by permutation (StatXact, CYTEL Software Corporation, Cambridge, Massachussetts). Comparison of several dose levels are made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected (Agresti et al. 1990); for individual intergroup comparisons (ie the positive control group) this procedure simplifies to a straightforward permutation test (Gibbons 1985). For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores are used, ie exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST: One female animal dosed with HP-10 at 2000 mg/kg had convulsions and died immediately after dosing. This death was considered to be due to mis-dosing. Results from the three remaining animals in this dose group showed that a dose level of 2000 mg/kg was tolerated, and this level was considered to be an appropriate maximum for use in the micronucleus test. Dose levels of 500, 1000 and 2000 mg/kg bodyweight were chosen for use in the main test.

MICRONUCLEUS TEST
Clinical signs and mortalities: No mortalities were obtained in the micronucleus test. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.
Micronucleated immature erythrocyte counts (mie): The test substance did not cause any statistically significant increases m the number of micronucleated immature erythrocytes at either sampling time (P>0.01).
Mitomycin C caused large, highly significant increases (P<0.001) in the frequency of micronucleated immature erythrocytes.
Micronncleated mature erythrocytes (mme): The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.
Proportion of immature erythrocytes (% ie/ie +me): The test substance failed to cause any significant decreases in the proportion of immature erythrocytes (P>0.01).
Mitomycin C did not cause any statistically significant decreases in the proportion (P>0.01) – it should be noted that even very cytotoxic compounds such as mitomycin C do not always produce a substantial decrease in this proportion as early as the 24 hour sampling time because of the lag caused by erythrocyte maturation.

TABLE 1 – Summary of results and statistical analysis

Sampling time

Treatment

Dose (mg/kg)

% ie/(ie+me) (mean)

Incidence mie (mean)

Incidence mme (group mean)

24 Hours

Vehicle control

-

43

0.9

0.7

HP-10

500

45

0.6

0.0

HP-10

1000

49

0.3

0.8

HP-10

2000

49

1.2

0.0

Mitomycin C

12

49

48.6***

1.5

48 Hours

Vehicle control

-

44

0.5

1.1

HP-10

2000

36

0.4

0.3

% ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes examined

mme: Number of micronucleated cells calculated per 2000 mature erythrocytes examined

Results of statistical analysis using the appropriate nonparametric method of analysis based on permutation (one-side probabilities):

*** P<0.001 (highly significant)

otherwise P>0.01 (not significant)

† Occasional apparent errors of ± 1% may occur due to rounding of values for presentation in the table.

 

TABLE 2 – Results for individual animals – 24 hour sampling time

Treatment

Dose (mg/kg)

Animal number

% ie/(ie+me)

Incidence mie

me

Incidence mme

Vehicle control

-

M 201

54

0

470

0

M 202

44

1

559

0

M 203

35

0

650

0

M 204

32

2

678

1

M 205

32

2

686

1

F 206

51

0

491

0

F 207

58

0

416

0

F 208

57

2

429

0

F 209

28

2

718

0

F 210

40

0

624

0

HP-10

500

M 211

46

0

540

0

M 212

39

0

613

0

M 213

44

1

558

0

M 214

39

0

624

0

M 215

36

0

662

0

F 216

55

2

448

0

F 217

35

1

648

0

F 218

41

1

590

0

F 219

43

1

572

0

F 220

71

0

288

0

HP-10

1000

M 221

43

2

575

1

M 222

54

0

458

0

M 223

58

0

417

1

M 224

50

0

503

0

M 225

38

0

618

0

F 226

52

0

476

0

F 227

68

1

328

0

F 228

28

0

724

0

F 229

52

0

477

0

F 230

49

0

510

0

HP-10

2000

M 231

53

4

468

0

M 232

38

0

620

0

M 233

50

0

497

0

M 234

48

2

516

0

M 235

48

0

523

0

F 236

50

2

500

0

F 237

34

2

656

0

F 238

46

0

541

0

F 239

72

2

272

0

F 240

54

0

465

0

Mitomycin C

12

M 241

59

32

433

0

M 242

50

54

507

0

M 243

61

47

393

1

M 244

35

35

738

0

M 245

42

58

584

0

F 246

58

60

422

0

F 247

49

25

508

1

F 248

47

30

531

2

F 249

35

66

654

0

F 250

59

79

415

0

%ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes

me: Total number of mature erythrocytes examined for micronuclei

mme: Number of mironucleated mature erythrocytes observed

 

TABLE 3 – Results for individual animals – 48 hour sampling time

Treatment

Dose (mg/kg)

Animal number

% ie/(ie+me)

Incidence mie

me

Incidence mme

Vehicle control

-

M 301

24

0

765

0

M 302

30

0

707

0

M 303

38

0

624

0

M 304

34

2

658

1

M 305

47

1

527

0

F 306

65

1

348

1

F 307

47

0

530

1

F 308

61

0

391

0

F 309

52

1

481

0

F 310

42

0

578

0

HP-10

2000

M 331

26

0

739

0

M 332

32

0

696

0

M 333

16

0

842

1

M 335

27

0

735

0

M 336

48

1

525

0

F 337

40

0

601

0

F 338

39

1

606

0

F 338

38

0

625

0

F 339

56

0

447

0

F 340

38

2

617

0

% ie/(ie+me): Proportion of immature erythrocytes

mie: Number of micronucleated cells observed per 2000 immature erythrocytes

me: Total number of mature erythrocytes examined for micronuclei

mme: Number of micronucleated mature erythrocytes

Conclusions:
Interpretation of results (migrated information): negative
No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with HP-10 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).
It is concluded that HP-10 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.
Executive summary:

The purpose of this study was to assess the potential of HP-10 to induce mutagenic effects in mice following acute intraperitonal administration using an in vivo cytogenetic system (Boller and Schmid 1970, MacGregor et a/1987, Mavoumin et al 1990). The intraperitoneal route was chosen for this particular study to maximise potential absorption of the test substance.

The procedures used were based on the recommendations of the following guidelines:

-OECD Guideline for the Testing of Chemicals. (1997) Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, Guideline 474.

-EEC Annex to directive 92/69/EEC (1992) Part B: Methods for determination of toxicity, B.12. Mutagenicity (Micronucleus test). Official Journal No. L 383 A, 35, 154.

-US EPA (1998) Health Effects Test Guidelines; OPPTS 870.5395 Mammalian erythrocyte micronucleus test. EPA 712-C-98-226.

 

This study was designed to assess the potential induction of micronuclei by HP-10 in bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was expected to be tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance and negative control were administered by intraperitoneal injection. The negative control group received the vehicle, corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with HP-10 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes (p<0.001).

It is concluded that HP-10 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.

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

Additional information

Additional information from genetic toxicity in vivo:

In Vitro Ames Assay:

Purpose of this test is an evaluation of the mutagenicity of 2,4,8,10-tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo [d.g] [1,3,2]dioxaphosphocin [other name: 2,2'-Methylenebis(4,6-di-tert-butylphenyl)2-ethylhexyl phosphitel by the microbial mutagenicity test.

2,4,8,10-Tetrakis(1,1-dimethyl ethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1,3,2] dioxaphosphocin did not induce revertant colonies more than twice of that of the solvent control on the S. typhimurium TA98, TA100, TA1535 and TA1537, and E. coli WP2uvrA with or without metabolic activation.

Positive control substances showed increases in revertant colony countmore than twice of the solvent control values with bacterial strains in various tests, and these suggested that susceptibilities of test bacterial strains were adequately maintained and the test was adequately carried out.

Based on the above results, the present test substance was assessed to be negative.

In Vitro Chromosome Abberation Test:

Chinese hamster lung cell line (CHL) was treated with 2,4,8,10-Tetrakis(1,1-dimethylethyl)-6-(2-ethylhexyloxy)-12H-dibenzo[d,g] [1,3,2] dioxaphosphocine in order to investigate the ability of the test substance to induce chromosomal aberrations.

The tests without metabolic activation (24 and 48 hours treatments) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml).

As a result, in the 48 hours treatment, 10.5% and 8.5% polyploidy induction were observed at the concentration levels of 4.0 and 5.0 mg/ml, respectively. Further, in the 24 hours treatment, 5.0%, 4.5%, 6.5%, 9.5% polyploidy induction were observed at the concentration levels of 2.0, 3.0, 4,0 and 5.0 mg/ml, respectively.

No structural aberration was observed at any concentration levels in both 24h and 48h treatments.

Tests with metabolic activation (6 hours treatment) were carried out at five different concentration levels (1.0, 2.0, 3.0, 4.0 and 5.0 mg/ml) in both -S9 and +S9 treatments. As a result, in the -S9 treatment, 7.0%, 7.0 % and 7.5 % polyploidy induction were observed at the concentration levels of 3.0, 4.0 and 5.0 mg/ml, respectively. Further, in the +S9 treatment, 5.0% polyploidy induction was observed at the concentration levels of 5.0 mg/ml. No structural aberration was observed at any concentration levels in both -S9 and + S9 treatment.

It is concluded that the test substance has the property of inducing polyploidy in CHL cells in the 48 hours treatment without metabolic activation.

This conclusion can be derived from the fact that the rate of appearance of the polyploid cells when applying the 48 hours treatment is more than 10%, which is the criterion for positive evaluation.

In Vivo Micronucleus Test:

This study was designed to assess the potential induction of micronuclei by HP-10 in bone marrow cells of mice. Mice were treated with a single intraperitoneal administration of the test substance at dose levels of 500, 1000 and 2000 mg/kg bodyweight. A preliminary toxicity test had previously shown that a dose of 2000 mg/kg (the standard limit dose for the micronucleus test) was expected to be tolerated; this level was therefore selected as an appropriate maximum for use in the micronucleus test.

The test substance and negative control were administered by intraperitoneal injection. The negative control group received the vehicle, corn oil. A positive control group was dosed orally, by intragastric gavage, with mitomycin C at 12 mg/kg bodyweight.

Bone marrow smears were obtained from five male and five female animals in the negative control, each of the test substance groups and the positive control group 24 hours after dosing. In addition bone marrow smears were obtained from five male and five female animals in the negative control and high level treatment groups 48 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature erythrocytes. The proportion of immature erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.

No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in mice treated with HP-10 and killed 24 or 48 hours later, compared to vehicle control values (p>0.01 in each case).

The positive control compound, mitomycin C, produced large, highly significant increases in the frequency of micronucleated immature erythrocytes (p<0.001).

It is concluded that HP-10 did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by intraperitoneal injection in this in vivo test procedure.

Justification for selection of genetic toxicity endpoint

Endpoint conclusion dervived using EU Method B12 and OECD guideline 474.

Justification for classification or non-classification

Based on the information avaiable the substance does not trigger and of the requirements for classification. Therefore the substance is Not Classified.