Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
Combined Micronucleus and Alkaline Comet Test in the Rat
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
testing proposal decision TPE-D-2114322496-49-01/F

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD474 combined with OECD489
Version / remarks:
Reviewed and agreed by the Laboratory Animal Welfare Officer and the Ethical Committee (DEC 14-45) as required by the Dutch Act on Animal Experimentation (February 1997).
GLP compliance:
yes
Type of assay:
other: combined in vivo micronucleus and in vivo COMET assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
recommended by international guidelines
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Strain:Crl:WI(Han)
- Age at study initiation: 6-9 weeks
- Weight at study initiation: 150-155 g
- Assigned to test groups randomly: yes
- Fasting period before study: limited supply of food the night before dosing (7 g/rat)
- Housing: 5 animals per sex in Macrolon cages (type MIV) with sawdust bedding and paper as enrichment
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.6 – 21.2°C
- Humidity (%): 40-70%
- Air changes (per hr): ca 10/hr
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
- Amount of vehicle (if gavage or dermal): no data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The substance was suspended in propylene glycol, specific gravity 1.036. The test formulation was kept on ice directly after weighing. The test item was sonicated (started at a low temperature and thereafter slowly increasing; range 3.0ºC- 24.5ºC for 16-64 minutes in the main assay) and a blender was (very shortly) used to get a homogeneous suspension.The formulation was clear yellow with small particles.

Treatment:
The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h and t=45 h, respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 (± 2 h) and t=45 (± 2 h). The animals were sacrificed by abdominal aorta bleeding under isoflurane anaesthesia at approximately t = 48 h.
Duration of treatment / exposure:
3 days
Frequency of treatment:
once daily
Post exposure period:
none
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
MTD as derived in pre-test
No. of animals per sex per dose:
5 males/dose (+ 3 additional animals dosed at 1000 mg/kg bw as replacement in case of mortality in the main group)
Males were selected based on a toxicity test at 500, 1000 and 2000 mg/kg bw where no substantial differences in toxicity between sexes were found.
Control animals:
yes, concurrent vehicle
Positive control(s):
For micronucleus part: cyclophosphamide
For Comet part: Ethyl Methanesulfonate

Examinations

Tissues and cell types examined:
Micronucleus test: bonemarrow
Comet Assay: liver, stomach and duodenum
Details of tissue and slide preparation:
Micronucleus test: bone marrow was isolated within 3 hours after third treatment from one femur, suspended in fetal calf serum and cetrifuged. Smears were prepared from the pellet that was resuspended in fetal calf serum, drops of this suspension were placed on the slid, air dreid and stained based on Giemsa (2 slides per animal). Micronuclei were scored in 4000 polychromatic erythrocytes and the ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes

Comet assay: liver, stomach and duodenum were isolated within 3 hours after third treatment, washed (stomach and duodenum), transferred in mincing buffer (stomach and duodenum) and minced on ice. For stomach and duodenum surface epithelia was gently scraped one time softly.
The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking waterbath at 37 °C for 21 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min).
The minced stomach and duodenum tissues were added to 10 mL of mincing buffer and incubated in a shaking waterbath at 37 °C for 15 minutes. Thereafter, a low centrifugation force was applied two times to remove undigested debris (40 g for 5 min). The supernatant was collected and filtered through a 100 µm Cell Strainer to purify the cell suspension. The purified cell suspension is centrifuged to precipitate the cells (359 g for 10 min).
Cell pellets of all tissues were suspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice. Three slides (pre-coated Comet slides) per animal were prepared in fresh alkaline solution and electrophoresis was performed at 4°C during 30 minutes.(1 Volt/cm for liver and 0.7 Volt/cm for stomach and duodenum). The slides were examined with a fluorescence microscope connected to a Comet Assay IV image analysis system. One hundred fifty Comets per slide were examined.
-Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
-Cells that showed overlap or were not sharp were not scored.
Evaluation criteria:
Micronucleus assay:
A test item is considered positive in the micronucleus test if:
a)At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b)Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative in the micronucleus test if:
a)None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b)All results are within the 95% control limits of the negative historical control data range.

Comet assay:
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a toxicologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.

A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.

Statistics:
Student's t-test, Dunnett test (ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany))

Results and discussion

Test resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
micronucleus test
Toxicity:
yes
Remarks:
at 500 mg/kg bw and above
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: abnormalities (large dark spots in the plasma of polychromatic erythrocytes) were observed in bone marrow slides at all doses
Key result
Sex:
male
Genotoxicity:
positive
Remarks:
dose dependent significant increase of tail intensity in liver, stomach and duodenum
Toxicity:
yes
Remarks:
at and above 500 mg/kg bw
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
1000 mg/kg bw: lethargy, dark yellow colored urine, ventral recumbency, ataxia, hairless skin was yellow colored, rough coat, hunched posture. One animal had a low body temperature and showed slow breathing. One animal from the main group died just prior isolation of the cells/tissues and was replaced by one of the additional animals. One of the additional animals also died approximately 3 hours after the third dose.
500 mg/kg bw: lethargy, dark yellow colored urine, ataxia
250 mg/kg bw: yellow colored urine

Any other information on results incl. tables

Micronucleus test

Treatment

Dose (mg/kg bw

Number of micronucleated polychromatic erythrocytes

(mean ± S.D.)(1,2)

Ratio polychromatic/normochromatic erythrocytes (mean ± S.D.)(1,3)

 

 

 

 

Vehicle control

0

3.8±1.6

0.95±0.04

substance

1000

3.2±1.3

0.85±0.22

substance

500

4.2±0.8

0.91±0.09

substance

250

4.2±0.8

1.00±0.10

CP

20

31.2±4.8(4)

0.43±0.17

Vehicle control = Propylene Glycol

CP = Cyclophosphamide.

(1)              Five animals per treatment group.

(2)              At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3)              The ratio was determined from at least the first 1000 erythrocytes counted.

(4)              Significantly different from corresponding control group (Students t test, p < 0.05).

 

Comet test

Liver

Tail Intensity (%)(1)

S.D.

Stomach

Tail Intensity (%)(1)

S.D.

Duodenum Tail Intensity (%)(1)

S.D.

Vehicle Control

8.13

1.21

13.19

8.86

26.69

13.07

V171629 250 mg/kg

30.48

10.20

46.62a

5.53

69.10a

15.21

V171629 500 mg/kg

19.80

22.77

41.32a

12.86

43.69

12.57

V171629 1000 mg/kg

38.93a

24.16

35.60a

19.52

59.30a

8.54

EMS 200 mg/kg

94.78***

1.26

93.30***

2.45

92.12***

1.42

(1) Five animals per treatment group.

***= p < 0.001(Student’sttest)
a= p < 0.05 Dunnett’s t test

Vehicle Control = Propylene glycol

EMS = Ethyl Methanesulfonate

 

Applicant's summary and conclusion

Conclusions:
It is concluded that the substance is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 1000 mg/kg (the maximum tolerated dose) under the experimental conditions described in this report. However, treatment with the substance caused bone marrow abnormalities (polychromatic erythrocytes with large dark spots in the plasma). Moreover, it is concluded that the substance shows a positive result (genotoxic effect) in the Comet assay in liver, stomach and duodenum post dosing of male rats up to a dose of 1000 mg/kg under the experimental conditions described in this summary.
Executive summary:

In a combined micronucleus and Comet assay, rats (5 males/group) received three daily doses at 0, 250, 500 and 1000 mg/kg bw of the substance in propylene glycol. Cyclophosphamide (MN) and ethylmethane sulphonate (Comet) were includeed as positive controls. At 500 and 1000 mg/kg bw a dose related increase of clinical signs was noted. At 1000 mg/kg bw one of the animals died on day 3. Preparation of bone marrow smears and concomitant scoring of mictonuclei showed that the substance is not clastogenic or aneugenic in the bone marrow.

Pre-coated Comet slides with stomach, duodenum and liver cells were subjected to electrophoresis and the tail intensity was dose related significantly increased in all three tissues. Therefore it was concluded that the substance shows a genotoxic effect.