Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD), but not on the test substance defined in Section 1.
Justification for type of information:
No genotoxicity data are available on S261a, but in vivo and in vitro studies on structurally related phthalates including DINP, BBP, and S278 provide useful information on the genotoxicity potential of S261a.
Cross-referenceopen allclose all
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD), but not on the test substance defined in Section 1.
Justification for type of information:
No genotoxicity data are available on S261a, but in vivo and in vitro studies on structurally related phthalates including DINP, BBP, and S278 provide useful information on the genotoxicity potential of S261a.
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: ca. 6-9 weeks
- Weight at study initiation: 17-35 g (although 17 g might include females used in another experiment)
- Assigned to test groups randomly: [no/yes, under following basis: ] no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: vehicle control used, but identity not described in publication
Details on exposure:
no data
Duration of treatment / exposure:
Two consecutive days
Frequency of treatment:
Daily
Post exposure period:
One day
Remarks:
Doses / Concentrations:
500
Basis:
actual ingested
mg/kg bw/day
Remarks:
Doses / Concentrations:
1000
Basis:
actual ingested
mg/kg bw/day
Remarks:
Doses / Concentrations:
2000
Basis:
actual ingested
mg/kg bw/day
No. of animals per sex per dose:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: presumably gavage
- Doses / concentrations: 20 mg/kg bw/day
Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No toxicity seen in a range-finding study at 2000 mg/kg bw/day.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treated on two consecutive days, and mice sacrificed on the third day.


DETAILS OF SLIDE PREPARATION:
Briefly, both femurs were removed from each treated mouse and the proximal ends were cut to expose the bone marrow, which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifugation and slides were prepared. After fixation in methanol, the slides were stained with acridine orange for about 1-2 minutes.

METHOD OF ANALYSIS:
The slides were evaluated at 400x by fluorescence microscopy. A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei.

OTHER:
Evaluation criteria:
no data
Statistics:
Statistical analysis included means and standard deviations of the micronucleus test data, and a test of equality of means was performed by standard one-way analysis of variance.
Sex:
male
Genotoxicity:
negative
Remarks:
at 2000 mg/kg bw/day
Toxicity:
no effects
Remarks:
at 2000 mg/kg bw/day
Vehicle controls validity:
not specified
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 2000 mg/kg bw/day (no details given of other dose levels)
- Solubility: no data
- Clinical signs of toxicity in test animals: no evidence of toxicity (extent of examination unclear from report)
- Evidence of cytotoxicity in tissue analyzed: no data


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Percentage PCEs with micronuclei was 0.22% for vehicle control, 0.14, 0.18 and 0.25% for DINP 1 at 500, 1000, and 2000 mg/kg bw/day, respectively, and 2.2% for cyclophosphamide at 20 mg/kg bw/day.
- Ratio of PCE/NCE (for Micronucleus assay): about 1:1 in all groups
- Statistical evaluation: No statistically significant increases in percentage of PCEs with micronuclei in DINP groups compared to vehicle control.
Conclusions:
Interpretation of results: negative
No in vivo genotoxicity data are available on S261a. Chromosome damage (increased frequency of micronuclei) was seen in the bone marrow erythrocytes of male mice (5/dose) given di(isononyl) phthalate (DINP 1; CAS 68515-48-0) by stomach tube on two consecutive days at up to 2000 mg/kg bw/day.
Executive summary:

No in vivo genotoxicity data are available on S261a. A micronucleus test was performed to assess the potential of di(isononyl) phthalate (DINP 1; CAS 68515-48-0) to induce chromosome aberrations in the bone marrow of mice.

 

Briefly, young male mice (5/dose) were administered DINP 1 by stomach tube at 0, 500, 1000, or 2000 mg/kg bw/day on two consecutive days. On the third day the animals were sacrificed, and slides were prepared from bone marrow erythrocytes. A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei in the treated group, and compared to that from the vehicle control group (no further details given on identity and dose of vehicle control) and the positive control group (receiving cyclophosphamide at 20 mg/kg bw/day).

 

Frequency of micronucleus formation was not elevated at any dose in the treated animals compared to the vehicle controls, and there was no evidence of toxicity (examination not described) at up to 2000 mg/kg bw/day. Treatment with the positive control induced a statistically significant increase in micronucleus formation, confirming the sensitivity of the testing procedure.

 

In conclusion, DINP 1 was inactive in an in vivo micronucleus test in which male mice (5/dose) were administered this phthalate by stomach tube at up to 2000 mg/kg bw/day on two consecutive days, and bone marrow erythrocytes were assessed for micronucleus formation on the third day.

Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Di(isononyl) phthalate (DINP) and di(isodecyl) phthalate (DIDP) are not mutagenic
Author:
McKee RH, Przygoda RT, Chirdon MA, Engelhardt G, Stanley M
Year:
2000
Bibliographic source:
J Appl Toxicol 20: 491-497

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DINP 1
- Molecular formula (if other than submission substance): C26H42O4
- Molecular weight (if other than submission substance): 420 [g/mol]
- Analytical purity: >99%
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: DINP 1 is produced from an alcohol feedstock that consists of roughly equivalent amounts of 3,4-, 4,6-, 3,5-, 4,5-, and 5,6-dimethylheptanol-1.
- Purity test date: no data
- Lot/batch No.: no data
- Expiration date of the lot/batch: no data
- Stability under test conditions: no data
- Storage condition of test material: no data

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage, MI
- Age at study initiation: ca. 6-9 weeks
- Weight at study initiation: 17-35 g (although 17 g might include females used in another experiment)
- Assigned to test groups randomly: [no/yes, under following basis: ] no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: vehicle control used, but identity not described in publication
Details on exposure:
no data
Duration of treatment / exposure:
Two consecutive days
Frequency of treatment:
Daily
Post exposure period:
One day
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500
Basis:
actual ingested
mg/kg bw/day
Remarks:
Doses / Concentrations:
1000
Basis:
actual ingested
mg/kg bw/day
Remarks:
Doses / Concentrations:
2000
Basis:
actual ingested
mg/kg bw/day
No. of animals per sex per dose:
5 males
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): no data
- Route of administration: presumably gavage
- Doses / concentrations: 20 mg/kg bw/day

Examinations

Tissues and cell types examined:
Erythrocytes from bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
No toxicity seen in a range-finding study at 2000 mg/kg bw/day.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Treated on two consecutive days, and mice sacrificed on the third day.


DETAILS OF SLIDE PREPARATION:
Briefly, both femurs were removed from each treated mouse and the proximal ends were cut to expose the bone marrow, which was then aspirated with fetal bovine serum into a centrifuge tube. The cells were collected by centrifugation and slides were prepared. After fixation in methanol, the slides were stained with acridine orange for about 1-2 minutes.

METHOD OF ANALYSIS:
The slides were evaluated at 400x by fluorescence microscopy. A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei.

OTHER:
Evaluation criteria:
no data
Statistics:
Statistical analysis included means and standard deviations of the micronucleus test data, and a test of equality of means was performed by standard one-way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
at 2000 mg/kg bw/day
Toxicity:
no effects
Remarks:
at 2000 mg/kg bw/day
Vehicle controls validity:
not specified
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: up to 2000 mg/kg bw/day (no details given of other dose levels)
- Solubility: no data
- Clinical signs of toxicity in test animals: no evidence of toxicity (extent of examination unclear from report)
- Evidence of cytotoxicity in tissue analyzed: no data


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Percentage PCEs with micronuclei was 0.22% for vehicle control, 0.14, 0.18 and 0.25% for DINP 1 at 500, 1000, and 2000 mg/kg bw/day, respectively, and 2.2% for cyclophosphamide at 20 mg/kg bw/day.
- Ratio of PCE/NCE (for Micronucleus assay): about 1:1 in all groups
- Statistical evaluation: No statistically significant increases in percentage of PCEs with micronuclei in DINP groups compared to vehicle control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
No in vivo genotoxicity data are available on S261a. Chromosome damage (increased frequency of micronuclei) was seen in the bone marrow erythrocytes of male mice (5/dose) given di(isononyl) phthalate (DINP 1; CAS 68515-48-0) by stomach tube on two consecutive days at up to 2000 mg/kg bw/day.
Executive summary:

No in vivo genotoxicity data are available on S261a. A micronucleus test was performed to assess the potential of di(isononyl) phthalate (DINP 1; CAS 68515-48-0) to induce chromosome aberrations in the bone marrow of mice.

 

Briefly, young male mice (5/dose) were administered DINP 1 by stomach tube at 0, 500, 1000, or 2000 mg/kg bw/day on two consecutive days. On the third day the animals were sacrificed, and slides were prepared from bone marrow erythrocytes. A total of 1000 erythrocytes were counted from each animal, and the total numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. A total of 1000 PCEs were evaluated for the presence of micronuclei in the treated group, and compared to that from the vehicle control group (no further details given on identity and dose of vehicle control) and the positive control group (receiving cyclophosphamide at 20 mg/kg bw/day).

 

Frequency of micronucleus formation was not elevated at any dose in the treated animals compared to the vehicle controls, and there was no evidence of toxicity (examination not described) at up to 2000 mg/kg bw/day. Treatment with the positive control induced a statistically significant increase in micronucleus formation, confirming the sensitivity of the testing procedure.

 

In conclusion, DINP 1 was inactive in an in vivo micronucleus test in which male mice (5/dose) were administered this phthalate by stomach tube at up to 2000 mg/kg bw/day on two consecutive days, and bone marrow erythrocytes were assessed for micronucleus formation on the third day.