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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets accepted scientific standard, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Lack of genotoxic activity of di(2-ethylhexyl) phthalate (DEHP) in rat and human hepatocytes.
Author:
Butterworth BE, Bermudez E, Smith-Oliver T, Earle L
Year:
1984
Bibliographic source:
Carcinogenesis 5, 1329-1335

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
not specified
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Purity>99.5%
- Source: Dr John R. Hodgson, Tenneco Chemicals, Saddle Brook, NJ or Eastman Chemical Products, Kingsport, TN

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: harles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: in hanging stainless steel mesh cages (Hazleton Systems Inc., Vienna, VA) in a mass air displacement room (Bioclean, Hazleton Systems Inc., Vienna, VA)
- Diet (e.g. ad libitum): ad libitum (NIH-07 open formula pelleted chow, Ziegler Bros., Gardner, PA
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 70+/-2°F (20-22°C)
- Humidity (%): 50+/-5
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Duration of treatment / exposure:
14 days (gavage) or 30 days (in diet)
Frequency of treatment:
daily (gavage) or ad libitum (diet)
Post exposure period:
2, 12, 24, 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
150, 500 mg/kg
Basis:
nominal conc.
by gavage
Remarks:
Doses / Concentrations:
12000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
at least 3 animals per group
Control animals:
yes, concurrent vehicle
Positive control(s):
DMN was given by gavage as a freshly prepared water solution.
2-AAF was given by gavage as a suspension in corn oil.

Examinations

Tissues and cell types examined:
Hepatocytes
Details of tissue and slide preparation:
Hepatocyte cultures were prepared at the times indicated after treatment.
After an attachment period of 1.5 h cultures were incubated with 10 µCi/ml [3H]thymidine (Amersham, Arlington Heights, IL) for 4 h followed by 14-16 h in medium containing no radioactivity.
Autoradiography was performed with a 14 day exposure period.
Evaluation criteria:
Silver grains over the nucleus minus the grains over an equal sized area in the cytoplasm was defined as net grains per nucleus (NG) and was quantitated with an automatic grain counter.
A negative number indicates there were more grains per unit area in the cytoplasm than in the nucleus. A cell with greater than 5 NG was considered in repair for both rat and human cells. One hundred and fifty cells were counted per data point for the in vitro rat studies and Cases 2 and 3 of the human study. Two hundred cells were counted per data point for Case 1 of the human study. Fifty cells were counted per slide, for three slides, for three animals (450 cells) for each data point for the in vivo experiments.
Cells in S-phase are easily recognized by the very dense labelling of silver grains over the nuclei. The percentage of tells in S-phase was determined for 1000 tells per slide for three slides, for three animals (9000 cells) per data point.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 1000 mg/kg the hepatocytes exhibited altered morphology and were badly clumped togethe
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No DNA repair was observed in male rat hepatocytes treated with 500 mg/kg DEHP at 2, 12, 24 or 48 h before sacrifice.
There was also no response with 1000 mg/kg at 12 h but the cells exhibited altered morphology and were badly clumped together; therefore, the 500 mg/kg dose was chosen for the remainder of the study.

There was also no response in male rats treated with 150 mg/kg/day DEHP for 14 consecutive days. No DNA damage as measured by strand breakage was seen in these animals. Hepatic peroxisomal proliferation was evident by electron microscopy and increased levels of hepatic palmitoyl CoA oxidase and carnitine acetyltransferase. There was also an increase in the liver to body weight ratio.

No induction of DNA repair or increased alkaline elution of DNA was seen in hepatocytes from female rats treated with 12 000 ppm DEHP in the diet for 30 days, 500 mg/kg DEHP at 2 h before sacrifice, or the combination of the two treatments.
Female rats treated with DEHP in the diet still produced a positive DNA repair response with 2-AAF. The alkaline elution response with the positive control 2-AAF was minimal and not nearly as great as that produced with DMN.
Hepatic peroxisomal proliferation had taken place in these animals as evidenced by electron microscopy and a large increase in palmitoyl CoA oxidase and carnitine acetyltransferase.
Although autoradiography did not demonstrate any DEHP induced DNA repair, DEHP did cause a 10-fold increase in the percentage of cells in S-phase in the male animals at the 24 h time point and in the animals given DEHP by gavage for 2 weeks. The percentage of hepatocytes in S-phase in the control female rats was higher than that seen in the male rats (Table IV). The female rats given DEHP in the diet showed only a doubling of the percent of cells in S-phase; however, the final percentage was greater than that observed in the treated males.

Any other information on results incl. tables

Response of DEHP in the in vivo-in vitro male rat hepatocyte DNA repair assay
Compound
Treatment Net nuclear grains (NG) ± SE a Percent of cells with > 5 NG ± SE b Percent of cells in S-phase ± SD e
               
DEHP d 500 mg/kg, 2 h e -5.3 ± 0.2 1 ± 0.3 0.01 ± 0.02
DEHP 500 mg/kg, 12 h -1.7 ± 0.5 8 ± 3 0.03 ± 0.0
DEHP 500 mg/kg, 24 h -5.4 ± 0.3 1 t 1   0.55 ± 0.29
DEHP 500 mg/kg, 48 h -3.1 ± 0.2 4 ± 2 0.01 ± 0.02
DEHP 150 mg/kg/day for 14 days f -5.6 ± 0.6 1 ± 0.4 0.50 ± 0.17
DEHP/DMN 150 mg/kg/DEHP for 14 days 18.1 ± 4.4 79 ± 9    
  10 mg/kg DMN at 2 h            
DMN 10 mg/kg, 2 h 59.2 ± 17.2 96 ± 2    
DMN 10 mg/kg, 12 h 38.9 ± 13.1 89 ± 5    
Corn oil 2 ml/kg, 2 h - 3.3 ± 0.9 5 ± 4 0.03 ± 0.06
Corn oil 2 ml/kg, 12 h -6.0 ± 0.5 1 ± 0.4 0.03 ± 0.03
Corn oil 2 ml/kg, 24 h -4.7 ± 0.2 0.3 ± 0.3 0.07 ± 0.09
a Standard errors shown represent animal to animal variation for at least three animais, three slides per animal, 50 cells per slide. The values for the 24 h corn oil and 12 h DMN are from two animais. The values for the 2 h corn oil are from one animal, SE is slide to slide variation. Results are consistent with historical positive and negative controls.
b Cells with ? 5 NG are considered in repair.
c Standard deviation shown is animal to animal variation for at least three animais, three slides per animal, 1000 cells per slide.
d DEHP was administered in corn oil by gavage at 1 ml/kg body weight. DMN was administered by gavage in water at 1 m1/kg body weight. 
e Time shown is the interval between dosing the animal and perfusing the liver.
f Last dose was given 2 h before liver perfusion.

Response of DEHP in the in vivo-in vitro female rat hepatocyte DNA repair assay
Compound Treatment Net nuclear grains (NG) f SE a Percent of cells with 5 NG ± Seb Percent of cells in S-phase± SD c
Control Control diet for 30 days -3.8 ± 0.6 3 ± 2 0.41 ± 0.20
DEHP 12 000 ppm DEHP diet for 30 days -3.0 ± 0.9 3 ± 2 0.90 ± 0.36
Control/DEHP Control diet for 50 days 500 mg/kg DEHP at 2 h d - 5.0 ± 1.2 3 ± 1 0.63 ± 0.67
DEHP/DEHP 12 000 p.p.m. DEHP diet for 30 days 500 mg/kg DEHP at 2 h -2.8 ± 0.6 3 ± 2 0.66 ± 0.39
Control/2-AAF Control diet for 30 days 50 mg/kg 2-AAF at 15 h 10.6 ± 2.2 73 ± 17    
DEHP/2-AAF 12 000 p.p.m. DEHP diet for 30 days 50 mg/kg 2-AAF at 15 h 6.2 ± 0.3 56 ± 5    
Control/2-AAF Cells from control animals in culture 20 h with 0.01 mM 2-AAF 43.5 ± 0.6 97 ± 1    
a Standard errors shown represent animal variation for three animais, three slides per animal, 50 cells per slide. Values for the in vivo 2-AAF are from one animal each and standard errors represent slide to slide variation. The in vitro 2-AAF is animal to animal variation from two animais.
bCells with >5 NG are considered in repair.
 c Standard deviation shown is animal to animal variation for three animais, three slides per animal, 1000 cells per slide.
d DEHP and 2-AAF were administered in corn oil by gavage at 2 ml/kg body weight. Time given is the interval between dosing the animal and perfusing the liver.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

The ability of DEHP to induce DNA damage or repair was examined in rat hepatocytes in vivo. Unscheduled DNA synthesis was measured by incorporation of [3H]thymidine into primary hepatocyte cultures immediately isolated from treated animals. DNA damage was measured by alkaline elution of cellular DNA from the same cultures.

In vivo-in vitro treatment regimens were: (i) female rats, 12 000 p.p.m. DEHP in the diet for 30 days; (ii) female rats, 12 000 p.p.m. in the diet for 30 days, followed by 500 mg/kg DEHP by gavage 2 h before sacrifice; (iii) male rats, 500 mg/kg DEHP by gavage 2, 12, 24, or 48 h before sacrifice; and (iv) male rats, 150 mg/kg/day by gavage for 14 days.

No chemically induced DNA damage or repair was observed in vivo in rat hepatocytes under any of the conditions employed. However, an increase in the percentage of cells in S-phase in the animals given DEHP was observed. These data indicate that DEHP does not exhibit ,direct genotoxic activity in the animals even with a treatment regimen which eventually produced tumors in a long term bioassay, and that both rat and human hepatocytes are similar in their lack of a genotoxic response to DEHP exposure in culture.