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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
Study period:
2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to an generally accepted test procedure

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000
Reference Type:
secondary source
Title:
O-Phenylphenol and its Sodium and Potassium Salts: A Toxicological Assessment
Author:
Bomhard, E. M. et al.
Year:
2002
Bibliographic source:
Crit. Rev. Toxicol. 32(6):551-626
Reference Type:
secondary source
Title:
Analysis of Genotoxicity and the Carcinogenic Mode of Action for Ortho-Phenylphenol
Author:
Brusick, D.
Year:
2005
Bibliographic source:
Environ Mol Mutagen 45:460-481

Materials and methods

Principles of method if other than guideline:
single cell gel/comet assay in rodents for detection of DNA damage, GLP study according to a test procedure scientifically acceptable
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
o-phenylphenol, purity: 99.8%
- Name of test material (as cited in study report): Preventol O extra, OPP, o-Phenylphenol
- Physical state: solid
- Analytical purity: 99.8%
- Purity test date: 29 June 1998, approved until 12 June 2000
- Lot/batch No.: E 0008
- Stability under test conditions: The test item was analysed to be stable in the vehicle at concentrations ranging from 25-200 mg/mL for at least 24 h.
- Storage condition of test material: at room temperature, protected from light

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 24-35 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study:
- Housing: individually in Makrolon type II cages with bedding of soft wood granules, type S 8/15 (Rettenmaier und Soehne, Fuellstoff-Fabriken, Ellwangen-Holzmuehle, Germany)
- Diet: Altromin 1324 Standard diet (Altromin GmbH, Lage, Germany), ad libitum
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23
- Humidity (%): 40-62
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): 2047001 (Henry Lamotte GmbH)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item and the positive control substance ere suspended in olive oil. For each animal the respective amount of test substance was sepataely suspende in a syringe.
Duration of treatment / exposure:
3, 8, and 24 h
Frequency of treatment:
single oral dose
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
250 and 2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
4 males per dose per treatment period
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; ethylmethanesulphonate
- Route of administration: oral (gavage)
- Doses / concentrations: 400 mg/kg bw
- Duration of treatment / exposure: 3 h

Examinations

Tissues and cell types examined:
Primary hepatocytes and kidney cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose selection was based on the publication of Sasaki et al. (1997) Mutat Res 395: 189-198

DETAILS OF SLIDE PREPARATION:
- Hepatocytes: After perfusion, incisions were made into each of the liver lobes. Subsequentially, liver cells were carefully collected with the help of a metal comp. The obtained cell suspension was filtered over fine gauze, filled up to 25 mL with cold WEI (Williams Medium E with 1% L-glutamine, 0.1% gentamycin sulfate, without foetal calf serum) and kept on ice for 5-10 min. Afterwards, the resulting supernatant was discarded and the pellet resuspended in 25 mL of cold WEI. Subsequent centrifugation occurred for 3 min at 50 x g and <15°C. Resuspending of the pellet in WEI and second centrifugation were performed as described above. The resulting pelet was resuspended in 5-15 mL WEI. An aliquot was used for the determination of cell viability and cell count by the method of trypan blue exclusion. The obtained viability value of the cell suspension after perfusion is a measure for the substance induced cytotoxicity during in vivo exposure.
- Kidney cells: Both kidneys were cut into half and the cortex region was isolated. The isolated tissue was cut into small pieces with a scalpel, suspended with a pipette and subsequently transferred to 50 mL of a digestion buffer. This buffer containes 25 mL collagenase solution and 25 mL trypsin solution (0.25% trypsin w/v in HBSS. Tissue pieces were incubated under stirring in a waterbath (37°C) for 20 min. After addition of 1 mL foetal calf serum (FCS) the cell suspension was filtered over gross gauze. Bovine serum albumine (10% w/v, 1 mL) was added and the cell suspension centrifuged at 68 x g and 4°C for 5 min. The pellet was resuspended in 25 mL cold HBSS and centrifuged again at 120 x g. The resulting pellet was resuspended in 10 mL cold HBSS: An aliquot was used for the determination of cell viability and cell count by the method of trypan blue exclusion. The obtained viability value of the cell suspension after perfusion is a measure for the substance induced cytotoxicity during in vivo exposure.

METHOD OF ANALYSIS:
The Comet Assay was performed according to the method of Singh et al. (1988) Exp. Cell Res. 175:184-191 with minor modifications. The different steps are comparable to the procedure described by Sasaki et al. (1997) Mutat Res 395: 189-198. Aliquots of the liver and kidney cell suspensions not greater than 100 µL were taken to reach an approximate cell number of 3-5E+04 liver cells and 8-10E+04 kidney cells, respectively. Cells were centrifuged at 180 x g for 6 min. The resulting pellet was mixed with 50 µL of 0.7% low melting agarose (LMA). The cell/LMA suspension was carefully pipetted onto slides already covered with two layers of 0.5% normal melting gaarose (NMA). Afterwards, a second LMA layer (0.7%, 50 µL) was placed on top. After lysis (1 h or overnight) buffer containing NA2EDTA, sacosinate, DMSO and freshly added Triton X-100; without proteinase K), alkaline treatment (20 min, NaOH and Na2EDTA, pH≥13) was performed followed by electrophoresis (60 min, 18 V, 300 mA) and neutralisation (Tris base, pH 7.5). Subsequently, slides were stained with ethidium bromide.
Evaluation of comets was performed using image analysis (Perceptive Instruments, Havehill, UK). Tail length, defined as distance between the middle of the head and the end of the tail, was used as assessment parameter. 50 cells/slide and 2 slides/animal were scored (100 cells total).
Evaluation criteria:
A response is considered positive, if a chemical induces a dose dependent mean increase of the tail length per dose group of more than 25% above the negative control group. Increases <25% compared to the negative control are considered negative. However, these criteria may be overruled by good scientific judgement.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
At 2000 mg/kg bw clinical signs of systemic toxicity was noted. 2/12 animals of that high dose died. No cytotoxicity was noted in hepatocytes and kidney cells of any of the animals of the treatment and control groups (positive and vehicle controls).
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Table 1.   Table for In Vivo Comet assay results of liver and kidney cells

Organ

Dose group

Tail length [µm, mean* ± SD]

3 h

8 h

24 h

Liver

Neg. control

34.17 ± 3.24

34.58 ± 2.33

34.77 ± 1.80

OPP

 

 

 

250 mg/kg

40.02 ± 0.84

32.63 ± 3.56

35.30 ± 4.51

2000 mg/kg

35.93 ± 3.70

31.98 ± 2.14

36.80 ± 3.33

EMS400 mg/kg

48.60 ± 4.58

48.72 ± 3.57

45.30 ± 1.87

Kidney

Neg. control

23.23 ± 1.99

23.48 ± 0.87

20.56 ± 0.45

OPP

 

 

 

250 mg/kg

20.93 ± 1.11

21.76 ± 1.02

21.65 ± 2.57

2000 mg/kg

19.71 ± 1.00

21.54 ± 1.35

23.27 ± 3.37

EMS400 mg/kg

34.60 ± 2.25

33.90 ± 2.11

32.75 ± 4.05

* 100 cells evaluated

 

Table 2.   Table for Cytogenetic In-Vivo-Test: Cytotoxicity

 

negative control

250

2000

positive control

Number of cells evaluated

100

Cytotoxicity: Liver cells

Sacrifice Time: 3h

Absolute Viabilitya

79.0±8.04

77.6±5.04

76.2±5.06

74.9±3.16

Relative Viabilityb[%]

100

98.1

96.4

94.8

Sacrifice Time: 8h

Absolute Viabilitya

81.0±5.48

77.0±2.27

73.5±1.30

78.1±4.68

Relative Viabilityb[%]

100

95.1

90.8

96.4

Sacrifice Time: 24h

Absolute Viabilitya

82.9±3.61

77.7±4.96

74.2±1.72

72.3±9.77

Relative Viabilityb[%]

100

93.7

89.5

87.2

Cytotoxicity: Kidney cells

Sacrifice Time: 3h

Absolute Viabilitya

92.3±1.56

91.6±2.758

86.2±6.37

89.9±2.53

Relative Viabilityb[%]

100

99.2

93.4

97.4

Sacrifice Time: 8h

Absolute Viabilitya

92.7±2.86

91.3±2.11

90.1±2.69

93.9±2.31

Relative Viabilityb[%]

100

98.5

97.2

101.3

Sacrifice Time: 24h

Absolute Viabilitya

92.5±1.63

92.5±1.73

90.0±1.87

93.3±2 43

Relative Viabilityb[%]

100

100

97.3

100.9

a= mean viability of cell preparation per dose group after perfusion
b= relative to negative control animals

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative