Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Sep - Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
September 26, 2014
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz
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
- Batch No.of test material: L85-142
- Expiration date of the Batch: 01 August 2017
- Purity: 73.7 % (tolerance +/- 1.0 %)
- Appearance: solid, white

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (2 - 8 °C), protected from light and moisture
- Solubility and stability of the test substance in the solvent: confirmed indirectly by dose formulation analytics

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test item was formulated in DMSO /PEG 400 (3 / 7). Sonicating and moderate heating (< 40 °C) of the formulation were used to formulate the test item. The different test item concentrations were prepared serially.

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

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recommended test system in corresponding guidelines.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 185.1 g (mean value, SD +/- 8.1 g)
- Assigned to test groups randomly: yes
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet (certified), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 45 - 65 % (except for deviations)
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO / polyethylene glycol 400 (3/7)
- Justification for choice of solvent/vehicle: The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation.
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw
- Purity: 30 % DMSO, 70 % PEG
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in DMSO / PEG 400 (3/7). Sonicating and moderate heating (< 40 °C) of the formulation were used to formulate the test item. The different test item concentrations were prepared serially. The preparations were made freshly before the dosing occasion.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
All animals received a single standard volume orally once.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Remarks:
high dose, 24 h and 48 h sampling time
Dose / conc.:
250 mg/kg bw (total dose)
Remarks:
medium dose, 24 h sampling time
Dose / conc.:
125 mg/kg bw (total dose)
Remarks:
low dose, 24 h sampling time
No. of animals per sex per dose:
7 animals per dose group
5 animals per vehicle and positive control group, respectively
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: orally once by gastric gavage
- Doses / concentrations: 20 mg/kg bw
- Volume administered: 10 mL/kg bw

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): The administered volume was 20 mL/kg bw each treatmet. Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum (FBS) using a syringe. The nucleated cells were separated from the erythrocytes by using the method of Romagna. Briefly, the cell suspensions were passed through a column consisting of α-Cellulose and Cellulose. The columns were then washed with Hank's buffered saline. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald / Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least per animal 4000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 500 erythrocytes. The analysis was performed with coded slides.
Evaluation criteria:
A test substance is classified as positive in the assay if
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95 % control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and / or further investigations.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes, applying above mentioned criteria, is considered non-mutagenic in this system, given that there is evidence for bone marrow exposure.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw.

The study is considered valid, as the following criteria are met:
1. The concurrent negative control is considered acceptable for addition to the laboratory historical control database (should ideally be within the 95 % control limits of the distribution of the historical negative control database).
2. At least 5 animals per group can be evaluated.
3. The appropriate number of doses and cells have been analysed.
4. PCE to total erythrocyte ratio should not be less than 20 % of the negative control.
5. The positive control shows a statistically significant increase of micronucleated PCEs compared to the negative control and is comparable to those in the historical positive control database.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 and 1000 mg/kg bw
- Solubility: The vehicle was chosen by the Sponsor and due to its relative non-toxicity for the animals and ability to formulate a suitable dosing preparation.
- Clinical signs of toxicity in test animals:
1000 mg/kg bw group: abdominal posture, sunken flanks, prophyrine strains, ruffled fur, slightly reduced spontaneous activity, apathy, salivation, disorientation, burrows itself in the bedding, ears hyperaemic, slight dyspnea
500 mg/kg bw group: abdominal posture, ruffled fur, slightly reduced spontaneous activity, salivation, diarrhea, lateral posture, thirsty
- Rationale for exposure: A preliminary study on acute toxicity was performed with two animals per sex and dose level under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
- Euthanization times: 24 h after exposure for 1000 mg/kg bw group, 48 h after exposure for 500 mg/kg bw group
- Other: No substantial differences between sexes in toxicity were observed. Thus, only male animals were used in the subsequent main experiment.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance tested as carbonate salt were below or equal to the value of the respective vehicle control group.
- Ratio of PCE/NCE (for Micronucleus assay): To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 500 erythrocytes.
- Appropriateness of dose levels and route: As estimated by two pre-experiments 500 mg/kg bw of the test substance tested as carbonate salt was suitable as highest treatment dose.
- Statistical evaluation: Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw.

Any other information on results incl. tables

Table 1: Results of pre-experiment

Clinical Symptoms

Hours post-treatment

0 – 1

2 – 4

5 – 6

24

30

48

1st Pre-experiment: 1000 mg/kg bw; 2 males / 2 females

Abdominal posture

2/2

0/1

1/2

The experiment was

stopped approx. 24 h

after application and the

remaining male animal

was humanely

euthanized

Sunken flanks

0/0

0/0

0/2

Porphyrine stains

0/0

0/0

0/2

Ruffled fur

0/0

0/0

0/2

Slightly reduced spontaneous activity

2/2

1/1

1/0

Apathy

0/0

0/1

0/2

Salivation

0/0

0/2

1/2

Disorientation

0/0

0/1

0/0

Burrows itself in the bedding

0/0

1/1

0/0

Ears hyperaemic

0/0

1/1

0/0

Slight dyspnea

0/0

0/0

0/2

Death

0/0

1*/0

0/0

0/2

2nd Pre-experiment: 500 mg/kg bw; 2 males / 2 females

Abdominal posture

0/0

2/1

1/1

0/0

0/0

0/0

Ruffled fur

0/0

0/0

0/0

2/0

0/0

0/0

Slightly reduced spontaneous activity

0/0

2/2

2/2

0/0

0/0

0/0

Salivation

0/0

1/1

0/0

0/0

0/0

0/0

Diarrhea

0/0

0/0

0/0

2/0

0/0

0/0

Lateral posture

0/0

0/0

1/1

0/0

0/0

0/0

Thirsty

0/0

0/0

2/2

0/0

0/0

0/0

* male No. 1 was euthanized in moribund condition (lateral posture, coma, salivation, hind legs and testis cyanotic)

Table 2: Clinical examination results of the main experiment

Clinical symptoms

Hours post-treatment (males)

0 - 1

2 - 4

5 - 6

24

48

High dose: 500 mg/kg bw (14 males at 1 to 24 h; 7 males at 48 h)

Abdominal posture

14

11

0

0

0

Partially closed eyes

0

5

0

0

0

Slightly reduced spontaneous activity

14

14

14

14

7

Salivation

14

14

0

0

0

Burrows itself in the bedding

14

0

0

0

0

Sleepy

14

0

0

0

0

Medium dose: 250 mg/kg bw (7 males)

Abdominal posture

7

7

0

0

Not applicable

Partially closed eyes

0

5

0

0

Slightly reduced spontaneous activity

7

7

4

2

Salivation

7

0

0

0

Burrows itself in the bedding

7

0

0

0

Sleepy

7

0

0

0

Low dose: 125 mg/kg bw (7 males)

Abdominal posture

3

5

0

0

Not applicable

Partially closed eyes

0

3

0

0

Slightly reduced spontaneous activity

7

0

0

0

Salivation

7

0

0

0

Burrows itself in the bedding

7

0

0

0

Sleepy

7

0

0

0

Table 3: Summary of Micronucles Test Results

Test group

Dose [mg/kg bw]

Sampling time

Mean MN/4000 PCE

SD MN/4000 PCE

Range

Ratio PCE/total Ery

% ratio Vehicle

Min

Max

Vehicle

0

24

11.4

5.8

6

20

0.573

100.00

Dose 1

125

24

8.3

2.6

5

13

0.562

98.08

Dose 2

250

24

9.0

3.6

4

12

0.584

101.92

Dose 3

500

24

8.6

2.9

6

13

0.575

100.35

Positive

20

24

102.6

39.7

49

154

0.527

91.97

Vehicle

0

48

15.2

5.8

10

25

0.575

100.00

Dose 3

500

48

14.0

3.5

8

18

0.545

94.78

MN: Micronuclei

SD: Standard Deviation

PCE: Polychromatic Erythrocytes

Ery: Erythrocytes

Table 4: Biometry

Negative control versus test group

Significance

p

Dose 1 – 125 mg/kg bw; 24 h

-

0.459

Dose 2 – 250 mg/kg bw; 24 h

-

0.362

Dose 3 – 500 mg/kg bw; 24 h

-

0.618

Positive control – 20 mg CPA/kg bw; 24 h

+

0.012

Dose 3 – 500 mg/kg bw; 48 h

-

0.870

-: not significant

+: significant

CPA: Cyclophosphamide

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item tested as carbonate salt did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the rat.
Executive summary:

This study was performed to investigate the potential of the test substance tested as carbonate salt to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the rat.

The test item was formulated in DMSO / PEG 400 (3/7), which was also used as vehicle control. The volume administered orally was 20 mL/kg bw. The volume of the positive control administered was 10 mL/kg bw.

24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei except for the negative and positive control groups with five animals each. At least 4000 polychromatic erythrocytes (PCEs) per animals were scored for micronuclei.

To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 500 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 125, 250, and 500 mg/kg bw.

48 h preparation interval: 500 mg/kg bw.

The animals treated with the vehicle control did not exhibit any clinical symptoms.

Clinical symptoms in the main experiment included abdominal posture, partially closed eyes, slightly reduced spontaneous activity, salivation, as well as sleepiness. The animals of all dose levels were affected.

The observed systemic toxicity at the tested doses is indicative for a systemic distribution of the test item. Thus, bioavailability of the test item under the tested conditions is assumed.

This was additionally confirmed by analytical detection of the test item in plasma.

The highest dose was estimated by two pre-experiments to be suitable.

A correction factor of 1.36 was applied based on test item purity data provided by the sponsor.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance tested as carbonate salt did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

20 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test item tested as carbonate salt did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the rat.

Therefore, the test substance tested as carbonate salt is considered to be non-mutagenic in this micronucleus assay.