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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb 2020 - 30 Apr 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 Feb 2020 - 30 Apr 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
29 July 2016.
Deviations:
yes
Remarks:
Only 3 or 4 animals were used for the isolation of the stomach in the control, low- and high-dose groups due to a technical error. All other values were within the historical controls and the results were clearly negative, so this did not impact the study
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material:
9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
In refrigerator (2 - 8 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Suspension.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 138 ± 8.4 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Not specified.
To: 09 Apr 2020.
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.
Duration of treatment / exposure:
Three consecutive days.
Frequency of treatment:
Daily.
Post exposure period:
Tissue samples taken 3 - 4 hours after administration of final dose.
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Maximum tolerable dose. Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg bw/day in a preliminary dose range finding study.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulphonate.
- Route of administration: Gavage.
- Doses / concentrations: 200 mg/kg bw, dissolved in physiological saline, administered twice.
Tissues and cell types examined:
Cells were isolated from the liver, glandular stomach, duodenum and kidney.
Details of tissue and slide preparation:
Minced liver or kidney tissue was added to collagenase and dissolved in HBSS (saline). This suspension was shaken and centrifuged. The cell pellet was resuspended in HBSS and kept on ice prior to preparation of the slides.

Tissue from the glandular stomach and duodenum was stored on ice in "mincing buffer incomplete" (HBSS + EDTA). The surface epithelium of both the glandular stomach and duodenum was discarded as it contains a high proportion of apoptotic cells which distort the comet analysis. The cells, suspended in the buffer, were filtered though a 100 µm cell strainer and stored on ice prior to preparation of the slides.

Low melting point agarose was added to the cell suspensions and layered on a comet slide, which was then incubated for 10 - 35 minutes in the refrigerator.

Slides were kept overnight in the refrigerator, immersed in pre-chilled lysis solution. After rinsing, the slides were placed in freshly-prepared alkaline solution; electrophoresis was performed for 20 minutes (stomach and duodenum) or 30 minutes (liver and kidney). Following another rinse, the slides were immersed in absolute ethanol and allowed to dry, before staining with SYBR Gold fluorescent dye.
Evaluation criteria:
A test item was considered positive if all of the following criteria were met:
a) at least one treatment group demonstrated a statistically significant increase in % tail intensity vs. control.
b) the increase was dose-related.
c) any of the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, the test item was considered negative. If the data precluded making a conclusion of clearly positive or negative, the result was concluded as equivocal.
Key result
Sex:
male
Genotoxicity:
ambiguous
Remarks:
Kidney: Statistically significant and dose-related (p < 0.001 for the trend) increase in tail intensity, but the mean % tail intensity within the 95% limits of the historical control data. See table below
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
Liver: no statistically significant increase in % tail intensity.
Toxicity:
not examined
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
Glandular stomach: no statistically significant increase in % tail intensity.
Toxicity:
not examined
Vehicle controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
Duodenum: no statistically significant increase in % tail intensity.
Toxicity:
not examined
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Upper 95% confidence limit of historical control data: 25.55% tail intensity.
Platinum was quantifiable in plasma samples from high-dose (1000 mg/kg/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3-4 hours after the third dose. Therefore it was confirmed that the animals were exposed to the test item. No test item was detected in the animals dosed with vehicle.

Comet results for kidney.

Dose % Tail Intensity S.D.
0 mg/kg bw/day (vehicle control) 3.52% ± 0.72%
250 mg/kg bw/day 7.56% ± 3.00%
500 mg/kg bw/day 14.56% ± 4.58%
1000 mg/kg bw/day 12.59% ± 6.09%
EMS (positive control) 79.86% ± 4.58%

Historical data Comet assay Negative control

Liver
Tail Intensity (%)

Males and Females

Duodenum
Tail Intensity (%)

Males and Females

Stomach
Tail Intensity (%)

Males and Females

Kidney
Tail Intensity (%)

Males and Females

Mean

1.96

3.06

2.45

12.10

SD

0.92

1.52

1.39

8.46

n

85

45

60

30

Lower control limit

(95% control limits)

0.27

-0.86

-1.07

-1.35

Upper control limit

(95% control limits)

3.65

6.97

5.96

25.55

SD = Standard deviation

n = Number of observations

 

Kidney: Historical control data from experiments performed in Feb 2012 – July 2019

Liver, Stomach, Duodenum: Historical control data from experiments performed in Jan 2018 – July 2019

Conclusions:
When tested in the comet assay, tetraammineplatinum dichloride did not induce an increase in DNA damage in the liver, glandular stomach or duodenum of rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days. A statistically significant and dose-related (p < 0.001 for the trend) increase in DNA damage was seen in kidney cells, but the mean % tail intensity fell within the 95% limits of the historical control data. As such, this finding was considered to be equivocal evidence of a genotoxic effect.
Executive summary:

The potential for tetraammineplatinum dichloride to cause DNA damage was evaluated in a study following OECD 489 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received two doses of EMS (200 mg/kg bw/day). Comet analyses were conducted on preparations of liver, glandular stomach, duodenum and kidney tissues.

There was no increase in % tail intensity in the liver, glandular stomach or duodenum, indicating that the test item is not genotoxic to these tissues.

There was a statistically significant and dose-related increase (p < 0.001) in DNA damage seen in the analysis of the kidney tissue. The tail intensity in animals dosed with 500 mg/kg bw/day was 14.56%, and in animals receiving 1000 mg/kg bw/day was 12.59%. However, these tail intensity values fell within the 95% confidence limits of the historical control data (upper limit 25.55%). As such, this finding was considered to be equivocal evidence of a genotoxic effect. No toxicity was observed in histopathological examination of the kidney tissues, indicating that this can be excluded as an indirect cause of the reported DNA damage.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report Date:
2020

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch number of test material: 9005305492.
- Expiration date of the lot/batch: 07 January 2021.
- Purity test date: CoA issued 26 November 2019.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2 - 8 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None.
- Final preparation of a solid: Test item was suspended in corn oil.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Suspension.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic items. Moreover, historical control background data has been generated with this strain.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks.
- Weight at study initiation: 138 ± 8.4 g (Mean body weight ± SD).
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Up to 5 animals of the same sex and in the same dosing group were housed together.
- Diet: Commercial pellets ad libitum, except during designated procedures.
- Water: Tap water, ad libitum.
- Acclimation period: At least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C.
- Humidity (%): 40 to 70%.
- Air changes (per hr): ≥ 10.
- Photoperiod: 12 hrs light/12 hrs dark, except during designated procedures.

IN-LIFE DATES:
From: Not specified.
To: 09 Apr 2020.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil.
- Source of vehicle: Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands.
Duration of treatment / exposure:
Three consecutive days.
Frequency of treatment:
Daily.
Post exposure period:
Tissue samples taken 3 - 4 hours after administration of final dose.
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Maximum tolerable dose. Mortality and severe toxicity were observed at doses of 1500 and 2000 mg/kg bw/day in a preliminary dose range finding study.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide.
- Route of administration: Gavage.
- Doses / concentrations: A single dose of 19 mg/kg bw, dissolved in physiological saline.

Examinations

Tissues and cell types examined:
Bone marrow from the femur.
Details of tissue and slide preparation:
The femurs were flushed with foetal calf serum and the cell suspension centrifuged. The supernatant was removed and a drop of the remaining cell suspension was spread across a clean slide and fixed with methanol. The slides were automatically stained with Giemsa using the Wright Stain Procedure.
Evaluation criteria:
The test item was considered positive if all of the following criteria were met:
a) at least one treatment group showed a statistically significant increase in frequency of micronucleated polychromatic erythrocytes.
b) the increase was dose related.
c) the results were outside the 95% confidence limits of the historical control data.

If none of the above criteria were met, the test item was considered negative.

The incidence of micronuclei was assessed in at least 4000 polychromatic erythrocytes per animal.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Platinum was quantifiable in plasma samples from high-dose (1000 mg/kg/day) satellite animals 1, 3, 6 and 12 hours after completing the second day of treatment. Moreover, platinum was quantifiable in plasma samples from all high-dose animals taken at necropsy approximately 3-4 hours after the third dose. Therefore it was confirmed that the animals were exposed to the test item. No test item was detected in the animals dosed with vehicle.
No statistically significant increase in the frequency of micronucleated polychromatic erythrocytes was observed. A slight increase was seen in all treatment groups that was within the 95% limits of the historical control data.

Treated animals showed no decrease in the PCE:NCE ratio, indicating a lack of toxicity to the bone marrow.

Applicant's summary and conclusion

Conclusions:
Tetraammineplatinum dichloride did not induce an increase in micronucleated polychromatic erythrocytes in rats administered up to 1000 mg/kg bw/day by gavage on three consecutive days.
Executive summary:

The in vivo clastogenicity of tetraammineplatinum dichloride, as evaluated by its ability to induce micronuclei in polychromatic erythrocytes, was assessed in a study following OECD 474 and according to GLP. Male Wistar rats (5/group) were given gavage doses of 250, 500 or 1000 mg/kg bw/day of the test item on three consecutive days, or a vehicle control. The concurrent positive control group received a single dose of cyclophosphamide. Bone marrow was harvested from the femurs and assessed for micronuclei.

There was a slight but not statistically significant increase in micronucleated polychromatic erythrocytes in all treatment groups, but the incidences fell within the 95% limits of the historical control data. On that basis, tetraammineplatinum dichloride was concluded to be non-genotoxic under the conditions of this assay.