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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records
Reference
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:
June 3 - September 25, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The reported data are reliable with restrictions; quality and stability of the tested substance are not mentioned. The study is performed under GLP conditions. The used methods are well documented and seem equivalent to OECD Guideline 473.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
quality and stability of test substance was not defined
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosomal aberrations
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
Range-finding: 0, 5 µg/ml - 5 mg/ml
Assay with activation: 0, 5, 10, 15 µg/ml
Assay without activation: 0, 2, 5, 10 , 15 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
other: at all time-points, except at 48 h. fixation
Positive control substance:
methylmethanesulfonate
Remarks:
at 48 hours positive control is lacking Migrated to IUCLID6: cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 18 - 24 hours
- Expression time (cells in growth medium): 6, 12, 24, 48 hours

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

NUMBER OF CELLS EVALUATED: 100 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; chromosomal aberrations
Evaluation criteria:
not mentioned.
Statistics:
Chi-square analysis and Dunnett's t-test were used to analyze the number of cells with structural aberrations and structural aberrations per cell, respectively. Results were considered statistically significant at the probability level of p < 0.05. Linear regression of linear and log dose versus response was used to analyze for dose-response relationship. A statistically significant probability level of p<0.05 was used.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
only at highest dose at t=48 hours
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Additional information on results:
RANGE-FINDING
Cytotoxicity was observed at 10 µglml, as indicated by a lengthening of average cell generation time to approximately 24 hrs and 18 hrs for treatment in the absence and presence of activation, respectively.

SCREENING STUDIES:
Without activation:
Santovar A levels of 2, 5, 10 and 15 µg/ml were chosen as the highest four scorable doses. However, after scoring and decoding, the 15 µg/ml treatment group was found not to contain enough scorable cells (2 or less). The results are therefore not reported. A decrease in mitotic index was observed at 5 µg/ml and higher doses for 6 hours and 10 µg/ml for 12 hours, indicating that cytotoxic levels were tested. Statistically significant increases in percentage cells with structural aberrations and in average structural aberrations per cell were observed at the 10 µg/ml treatment level of Santovar A at the 48 hour harvest time. The aberrations were chromatid deletions and interchanges, and 3 chromosome-type exchanges. However, this response was entirely a factor of the high dose point. No increases were observed at any other lower dose levels.

With activation:
The Santovar A levels of 5, 10 and 15 µg/ml were selected as the three highest scorable doses. A decrease in mitotic index was observed at 15 µg/ml for 6 hours. A statistically significant increase in structural aberrations per cell was observed for the 10 µg/ml treatment group at the 6 hour harvest times. Since 92 metaphases were scored for this dose and all of the structural aberrations were found in one cell, this response was evaluated not to be a test-chemical related effect. This evaluation is supported by the negative responses observed at the other harvest times. No statistically significant dose-response relationship was observed at any harvest times.
Conclusions:
Interpretation of results (migrated information):
negative

The results of the chromosomal aberration assay in CHO cells suggest a negative tendency for 2,5-Di-t-pentylhydroquinone for inducing chromosomal aberrations in experiments with and without S9-activation during various incubation periods.
Executive summary:

Santovar A was tested for its potential to induce chromosomal aberrations in cultured Chinese hamster ovary (CHO) cells. CHO cells were treated for 5 hours with 1, 2, 5, 10 and 15 μg/ml of the test chemical. The dose levels 2, 5, and 10 μg/ml were scored and reported in the absence of exogenous activation and 5, 10 and 15 μg/ml in the presence of exogenous activation (Aroclor 1254 induced rat liver homogenate). The cells were harvested at 6, 12, 24 and 48 hours after initiation of treatment. The harvest times were calculated for each treatment condition to allow the detection of effects of treatment at different cell cycle stages. In the non-activated study, statistically significant increases in percentage cells with structural aberrations and structural aberrations per cell were observed only for 10 μg/ml Sansovar A at the 48 hr harvest time. A statistically significant dose- response relationship was also observed at this harvest time. No statistically significant increases were observed for Santovar A treatment with activation. Under these conditions, Santovar A was concluded to be negative in the presence of metabolic activation. In the absence of activation, Santovar A was concluded to be a possible clastogen. However, because of the single dose level and time point of the response, and that the response was not observed in the presence of activation, the clastogenicity may not be relevant to the in vivo situation.

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

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 13- December 21, 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is performed according to the applicable OECD and EPA guidelines under GLP conditions. No deviations were reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5385 (In Vivo Mammalian Cytogenetics Tests: Bone Marrow Chromosomal Analysis)
Deviations:
no
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Massachusetts
- Age at study initiation: 6.5 weeks
- Weight at study initiation: males: 163 - 200 g; females: 126 - 153 g.
- Assigned to test groups randomly: yes
- Fasting period before study: 18 hours
- Housing: individually in stainless, steel cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 3 days
Route of administration:
oral: unspecified
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Duration of treatment / exposure:
6, 18, 30 hours
Frequency of treatment:
single dose
Remarks:
Doses / Concentrations:
0, 1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw
Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION:
Cells were resuspended in a small amount of freshly prepared fixative. Slides were prepared by dropping the cell suspension on precleaned methanol-wet glass slides followed by flaming. Slides were stained in 3% Giemsa in distilled water for 10 minutes, rinsed in distilled water and air dried. Slides were cleared in xylene and coverslips mounted with Permount.
Evaluation criteria:
Cytogenetic abnormalities were classified on a standard scoring sheet according to chromosome or chromatid aberrations and further according to type of aberration. Aberrations were classified according to the nomenclature of Buckton and Evans, 1973 and Savage, 1975.
Statistics:
Mean number of aberrations per cell per rat (50 cells per rat) were analyzed for statistically significant increases in chromosome aberration by a one way analysis of variance (ANOVA). Each sampling time was analyzed separately as compared to its concurrent vehicle control group. The mean and standard deviation of aberratlons/cell were also determlned for each group of rats (500 cells: 50 cells per rat). The number of aberrant metaphases was analyzed by Chi-square analysis for statistically significant increases. Vehicle control groups were also analyzed by a one way ANOVA for time related differences in aberrations, as were the three groups of rats administered Santovar A. Statistical signlficance was determined at the p<= 0.05 probabillty level.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: rats expressed toxicity signs at 950 mg/kg bw, 1000 mg/kg bw was chosen for definitive study

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): no structural chromosomal aberrations induced by testing substance
- Appropriateness of dose levels and route: all rats dosed with 1000 mg/kg exhibited mild to severe toxicity signs at all times evaluated and 2 females at 18 hours testing group died. Thus, the tested substance was dosed near the maximum tolerated dose.
Conclusions:
Interpretation of results (migrated information): negative
Male and female Fisher 344 rats received 0 and 1000 mg/kg Santovar A by oral gavage. The hemopoietic cells of the bone marrow were analyzed after 6, 18, and 30 hours. No statistically significant increases in the incidence of aberrations or in the number of cells with one or more aberrations were observed in animals treated with Santovar A at 1000 mg/kg at any of the three sampling times evaluated. Therefore, under the conditions of this assay, Santovar A was not clastogenic to the hemopoietic cells of the rat bone marrow.
Executive summary:

This study was designed to evaluate the potential of Santovar A to induce structural chromosomal aberrations in the hemopoietic cells of the rat bone marrow when administered by oral gavage. Based on a preliminary study in which Santovar A was evaluated at 950 mg/kg, a dose of 1000 mg/kg of body weight was selected for evaluation at the 6, 18 and 30 hour sacrifice intervals. Vehicle control nice administered corn oil were included at each sampling time. The positive control, cyclophosphamide (CP) at 50 mg/kg was included at the 18 hour sampling time. Santovar A was administered in single oral doses to three groups of fasted young adult Sprague Dawley rats. Approximately two hours prior to each sacrifice time, animals were administered colchicine at 4 mg/kg of body weight to arrest cells in metaphase. At the appropriate time, animals were sacrificed and both femurs were removed from each animal ad metaphase slides prepared. Slides were stained, coded and scored for chromosomal aberrations.

All rats dose with Santovar A (1000 mg/kg) exhibited from mild to severe pharmacotoxic signs. In addition, to females from the 18 hours harvest group died. These results suggest that Santovar A was tested at or near the maximum tolerated dose.

A total of 50 metaphase cells were analysed for each animal (when possible) for the presence of chromatid and chromosome type aberrations. Aberrations were classified according to type on a standard scoring sheet and the number of aberrations in each cell tabulated. The number of centromeres in each cell was counted and recorded.

Data was evaluated for statistically significant increases in aberrations per cell in treatment groups as compared to the vehicle control groups. The proportion of aberrant metaphases was also evaluated for statistically significant increases over the vehicle control groups. Data were evaluated separately for each harvest interval.

The positive control article, CP, resulted in significant increases in the incidence of chromosome aberrations and in the proportion of metaphases with one or more aberrations.

No statistically significant increase in the incidence of aberrations or in the number of cells with one or more aberrations were observed in animals treated with Santovar A (1000 mg/kg) at any of the three sampling times evaluated. Therefore, under the conditions of this assay, Santovar A was not clastogenic to the hemopoietic cells of the rat bone marrow.

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

Additional information

2,5 -Di-tert-pentylhydroquinone showed a negative tendency in three in vitro mutagenicity tests; in one other in vitro mutagenicity test, 2,5 -Di-tert-pentylhydroquinone showed an ambiguous to mild positive response.

In vivo, the mutagenicity of 2,5 -di-tert-butylhydroquinone was studied in male and female rats. Upon oral gavage of sublethal doses, bone marrow cells were analysed after 6, 18, and 30 hours. No statistically significant increases in the incidence of aberrations or in the number of cells with one or more aberrations was observed. Thus, 2,5 -di-tert-butylhydroquinone was not clastogenic under the circumstances as described above.

Short description of key information:

In vitro genotoxicity of 2,5-Di-t-pentylhydroquinone was tested in various cell systems. In four key studies the experimental results of 2,5-Di-t-pentylhydroquinone in a chromosomal aberration assay, a mammalian cell gene mutation assay, a DNA damage and repair assay, and in cultured E. Coli and S. Typhimurium cells (Ames test) are described.

In vivo genotoxicity was studied by administration of 2,5-Di-t-pentylhydroquinone to male and female rats and subsequent analysis of bone marrow cells for chromosomal events.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the above mentioned results, the substance does not need to be classified according to the Dangerous Substance Directive 67/548/EC and CLP Regulation (EC) 1272/2008.