Ibuprofen

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic in an Ames assay up to a concentration of 5000 µg/plate.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

see "Principles of method other than guideline"

Principles of method if other than guideline:

Salmonella strains TA 97a, TA 100, TA 102 have been used; the (additional recommended) Salmonella strains TA 1535 and TA98 were absent. Additionally, two experiments were performed with 2 replications for each concentration in stead of 1 experiment with triplicate plating.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purchased from Sigma Chemical Company (St. Louis, MO).

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA97a, TA100, TA102

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital induced S9 rat liver homogenate

Test concentrations with justification for top dose:

- Test concentrations: 1, 10, 100, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylenediamine; 2-aminofluorene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 experiments performed, 2 replications per experiment (4 total)

Evaluation criteria:

The revertant colonies on all the plates were counted. Statistical calculations were carried out with the mean revertant colonies of each concentration of the each independent treatment group with respective controls

Statistics:

Dunnett’s multiple comparison

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- TA 97a and TA100 without metabolic activation: mean number of revertant colonies per plate were significantly higher at concentrations of 1 and 10 µg/plate in the first experiment, but not in the second experiment. Also, no dose-response was observed.
- TA97a with metabolic activation: mean number of revertant colonies per plate were significantly higher at concentrations of 1, 10, and 100 µg/plate in the second experiment, but not in the first experiment. Also, no dose-response was observed.

The registrant of the test substance noted that the observed increase was relatively small and only statistics were used to determined whether a positive response was observed. Current procedure is that a test substance is considered to be positive in a bacterial gene mutation test if the mean number of revertant colonies on the test plates is increased in a dose-related manner or if a two-fold and/or greater increase was observed compared to the negative control plates. As this is not the case with any of the strains tested, it can be concluded that the test substance is not mutagenic in a bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In single studies, the test substance caused in vivo in mice increased sister chromatid exchanges at doses of 50 & 100 mg/kg bw (intraperitoneal) and 270 mg/kg (oral) and chromosmal abbrations were observed in bone marrow cells of mice at doses of 40 and 60 mg/kg body weight. In addition, from a secondary, regulatory source there is information

from a pharmaceutical manufacturer that

Ibuprofen did not show clinically relevant indications of mutagenic effects in in vitro and in vivo investigations.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: 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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A sister chromatid exchange (SCE) assay was performed in Swiss mice. Paraffin-coated BrdU tablets were implanted subcutaneous, and the test substance was administered once intraperitoneal (25, 50, 100 mg/kg, 1h after tablet implantation) or once orally (gavage, 270 mg/kg, 0.5 h after tablet implantation) in 5 male mice per dose. Twenty-four hours after implantation, bone marrow slides were prepared and stained with fluorescence-plus-Giemsa technique. Per dose 150 cells were counted. 

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Specific details on test material used for the study:

Ibuprofen, purchased from the Sigma Chemical Company (St. Louis, MO).

Species:

mouse

Strain:

Swiss

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Division of Laboratory Animals, Central Drug Research Institute, Lucknow.
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 30g
- Housing: 5 per cage with husk bedding
- Diet: standard rodent pellet diet (Gold Mohar, Lipton India Ltd., Chandigarh, India), ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2
- Humidity (%): 60 ± 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: intraperitoneal and oral (gavage)

Vehicle:

VEHICLES
- Intraperitoneal: DMSO
- Oral: distilled water in 2% gum acacia

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Intraperitoneal: injection of 75 µL/mouse
- Oral: 0.3 mL/mouse

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Single administration

Dose / conc.:

25 mg/kg bw/day

Remarks:

intraperitoneal

Dose / conc.:

50 mg/kg bw/day

Remarks:

intraperitoneal

Dose / conc.:

100 mg/kg bw/day

Remarks:

intraperitoneal

Dose / conc.:

270 mg/kg bw/day

Remarks:

oral

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Intraperitoneal: Mitomycin C, 1.5 mg/kg bw
- Oral: Cyclophosphamide in distlled water, 10 mg/kg

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dose selected for the in vivo studies of three drugs (ibuprofen, naproxen, ketoprofen) was based on the LD50 dose of ibuprofen available in the literature. The highest dose selected in the i.p. study (100 mg/kg) was approximately one-third of the LD50 (320 mg/kg) of mice for ibuprofen reported earlier.

TREATMENT AND SAMPLING TIMES: BrdU tablets were implanted subcutaneously in the flank of mice under ether anesthesia. The substance was administered intraperitoneal (1h after implantation) and oral (0.5 hour after implantation).

DETAILS OF SLIDE PREPARATION:
- I.P. For SCE analysis, colchicine 4 mg/kg was injected i.p. 22 h after Brdu tablet implantation. Two hours later bone marrow was expelled with 0.075 M KCl at 37 °C for 20 min, cells were fixed three times with methanol/acetic acid (3:1). The slides were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa technique.
- ORAL: Twenty-two hours after the BrdU tablet implantation, colchicine was injected, and the rest of the procedure was the same as described above.

METHOD OF ANALYSIS: All the slides were coded and 30 second-division metaphase cells (40 ± 2 chromosomes) per animal were scored for SCE frequencies, i.e. a total of 150 cells were scored per dose tested.

Statistics:

- Dunnett’s multiple comparison for i.p. SCE results
- Student's t-test to compare the oral SCR results of each treated group

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

at 50- and 100- mg/kg doses (intraperitoneal) and at 270 mg/kg (oral)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A significant increase in SCE was observed at 50- and 100-mg/kg doses. A single oral dose of ibuprofen (270 mg/kg) also gave a weak, but significant, increase in SCE when compared with control.
The SCE/cell after i.p. administration was 4.50, 5.04, 5.70, and 6.00 at 0, 25, 50, and 100 mg/kg bw.
The SCE/cell after oral administration was 4.68 and 6.16 at 0 and 270 mg/kg bw.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

mice were exposed for 2 weeks instead of two seperate single treatments; Limitations: no information on the sex of mice and environmental conditions and no specified information on oral administration

GLP compliance:

no

Type of assay:

other: chromosomal aberration test

Specific details on test material used for the study:

Test substance was received from ACME Pharmaceuticals Ltd. (Gujarat, India).

Species:

mouse

Strain:

Swiss

Sex:

not specified

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: institutional animal house of Shri Sarvajanik Pharmacy College (Gujarat, India)
- Age at study initiation: 7-9 weeks
- Diet: commercial mouse pellets; available ad libitum
- Water: available ad libitum
- Acclimation period: 7 days

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: suspension of the test material was prepared in 0.5% carboxymethyl cellulose

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

10 mg/kg bw/day

Dose / conc.:

20 mg/kg bw/day

Dose / conc.:

40 mg/kg bw/day

Dose / conc.:

60 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg), adminstered intraperitoneally (i.p.) 24 hours before termination.

Tissues and cell types examined:

The Mitotic Index and Chromosomal abberations were determined in bone marrow cells.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose selection was based on the recommended therapeutic doses of drugs for humans. Because the recommended oral dose of ibuprofen is 800–1,200 mg/day, the four dose levels selected were (10, 20, 40, and 60 mg/kg) the lower, middle, and higher of the human prophylactic doses, and one highest dose was also selected to be certain of a dose response.

DETAILS OF SLIDE PREPARATION: Animals were given 0.4 mL of 0.05% colchicine i.p. 90 minutes before sacrifice to stop the mitotic process in metaphase. At the time of death, both femurs were dissected out, bone marrow was extracted in 0.075 M of KCl, and the cell suspension was incubated for 20 minutes at 37°C. Cells were collected by centrifugation at 1,000 rpm for 10 minutes and were fixed three times with methanol/acetic acid (3:1). Chromosome slides were prepared by dropping the cell suspension onto clean chilled slides, then flame-dried, coded, and stained in dilute Giemsa solution.

METHOD OF ANALYSIS: Microscopic observations were performed with a magnification of 100× oil immersion. One hundred well-spread metaphases were scored per animal (500 metaphases per treatment group) at random. All aberrations, such as chromatid gaps, chromosomal gaps, chromatid breaks, chromosomal breaks, deletion, ring, dicentric ring, exchange, stickiness, acentrics, and fragmentation were considered equal, regardless of the number of breakages involved. Chromosomal aberrations per cell (CAs/cell) were calculated, including and excluding gapsMitotic index (MI) was calculated from 1,000 cells/animal and is expressed in percentage.

Statistics:

One-way analysis of variance followed by Dunnet's test using Prism software (Prism, 1997; Prism Software India Ltd., Nagpur, India) as an posteriori test.

Key result

Sex:

not specified

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A dose-related decrease in the mitotic index (%) was found: 8.200, 8.140, 7.500, 5.620*, and 5.060* for 0, 10, 20, 40, and 60 mg/kg bw, respectively
An increase in chromosomal aberrations per cell was found including and excluding cells with gaps: 0.036, 0.066, 0.122*, 0.308*, 0.416* (with gap), and 0.028, 0.058, 0.108, 0.288*, 0.366* for 0, 10, 20, 40, and 60 mg/kg bw, respectively. According to the authors further studies are requested to verify these results.

*Statistically significant

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

In a published study (Phillipose 1997) an Ames mutagenicity assay was performed, of which the methods were comparable to OECD guideline 471 with acceptable restrictions. Mutagenicity of the test substance was assessed by incubating the Salmonella strains TA 97a, TA 100 and TA102, with and without metabolic activation, according the plate incorporation method. Bacteria were exposed to concentrations of 1, 10, 100, 1000 and 5000 µg/plate in two separate experiments (2 plates per experiment). Cytotoxicity was observed at the highest concentration in all experiments. In the first experiment, a significant increase in revertant colonies was observed at concentrations of 1 and 10 µg/plate for Salmonella strains 97a (1st experiment without metabolic activation; 2nd experiment with metabolic activation) and T100 (1st experiment without metabolic activation). These small significant increases were only seen in 1 of the 2 experiments and no dose-response relation was observed. The registrant of the test substance also noted that the observed increase was relatively small and only statistics were used to determine whether a positive response was observed. Current procedure is that a test substance is considered to be positive in a bacterial gene mutation test if the mean number of revertant colonies on the test plates is increased in a dose-related manner or if a two-fold and/or greater increase was observed compared to the negative control plates. As this is not the case with any of the strains tested, it can be concluded that the test substance is not mutagenic in this bacterial reverse mutation assay.

Similarly in an older publication (Oldham 1986), the in vitro genetic toxicity of the test substance was evaluated in an Ames test carried out in accordance with Ames BN et al (1975). Strains tested were Salmonella typhimurium TA98, TA100, TA1535, TA1537, TA1538 at concentrations of 1, 10, 100, 500, 750, 1000 µg/plate both with and without the presence of a metabolic activation system (S-9). Two plates per dose for test substance and positive controls were used. The test substance was not mutagenic under the conditions of this test. There was no appreciable increase in the revertant colony counts induced by treatment with the test substance in any strain under any of die metabolic conditions. The spontaneous reversion frequencies (DMSO, negative controls) were consistent with historical data for all five tester strains. The positive control substances were strongly mutagenic under the respective conditions. Microscopic precipitation in die top agar was seen 750 µg/plate and more. Toxicity of the test substance was seen under all four metabolic conditions at doses >= 750 µg/plate, indicated by a thin lawn of bacterial growth and a decrease in the revertant colony count in all strains.

Animal data

In a published in vivo mammalian somatic cell study (Philipose 1997), a sister chromatid exchange (SCE) assay was performed in Swiss mice. Paraffin-coated BrdU tablets were implanted subcutaneous, and the test substance was administered once intraperitoneal (25, 50, 100 mg/kg, 1h after tablet implantation) or once orally (gavage, 270 mg/kg, 0.5 h after tablet implantation) in 5 male mice per dose. Twenty-four hours after implantation, bone marrow slides were prepared and stained with fluorescence-plus-Giemsa technique. Per dose 150 cells were counted. The number of SCEs were significantly increased at the doses of 50- and 100- mg/kg doses (intraperitoneal) and at 270 mg/kg (oral).

 

In a study performed according to OECD guideline 475 (Tripathi 2012), genotoxicity of the test material was determined in a chromosome aberration test by exposing Swiss mice to doses of 10, 20, 40, and 60 mg/kg body weight test substance, with 5 animals per dose. The test material was administered orally daily for a period of 14 days. After, bone marrow cells were isolated and the mitotic index and chromosomal aberrations were determined (1000 cells/animal and 100 metaphases per animal, respectively). A dose-related decrease in the mitotic index and an increase in chromosomal aberrations per cell was found in bone marrow cells. A statistically significant reduction in MI and an increase in CAs/cell were found for both the higher doses. According to the authors further studies are requested to verify these results.

Human data

Genetic toxicity was assessed in a sister chromatid exchange study, performed with cultured human T-lymphocytes (Ozkul, 1996; chapter 7.10.5). A total of 8 patients were given 800 mg/day of the test substance for a period of 2 weeks. The average number of sister chromatid exchanges in cultured lymphocytes from the patients, before and after treatment with the test substance did not differ significantly.

Conclusion

In conclusion, the test substance was not mutagenic in an Ames test. In an SCE assay and a chromosmal aberration test both performed with Swiss mice positive results were reported. Humans that were exposed did however not show an increase in SCEs. There is secondary information available from pharmaceutical manufacturers that Ibuprofen did not show clinically relevant indications of mutagenic effects in in vitro and in vivo investigations.

Justification for classification or non-classification

In single studies with mice, Sister Chromatid Exchanges (SCEs) and chromosome aberrations were reported. However, in a human study no increase in SCEs was found. The specialist information of a pharmaceutical manufacturer reported no clinically relevant indications of a mutagenic potential. The identified long term carcinogenicity studies (BASF 1972 and BASF 1976) on both rat and mice did not show any sign of carcinogenic properties of the test material. As genetic toxicity in the absence of carcinogenicity is unlikely, classification for genetic toxicity is considered not possible in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.