Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: chromosome mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
CAT-Acid
IUPAC Name:
CAT-Acid
Details on test material:
- Name of test material (as cited in study report): CAT-Acid
- Analytical purity: 99.7%
- Lot/batch No.: AD 91121201
- Expiration date of the lot/batch: August 02, 2013

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: bioanalytical experiment (non GLP) before 1st treatment:
mean value males 38.5 g (SD ¿ 2.6 g)
mean value females 32.3 g (SD ¿ 1.6 g)
bioanalytical experiment (non GLP) before 2nd treatment:
mean value males 37.8 g (SD ¿ 2.0 g)
mean value females 31.1 g (SD ¿ 1.5 g)
mutagenicity experiment before 1st treatment:
mean value males 33.9 g (SD ¿ 1.6 g)
mutagenicity experiment before 2nd treatment:
mean value males 34.2 g (SD ¿ 1.6 g)
- Assigned to test groups randomly: yes
- Housing: single
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 35 - 65 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.


IN-LIFE DATES: From: 2011-11-16 To: 2012-05-15

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Identity: corn oil
Supplier: Sigma-Aldrich Vertriebs GmbH
Catalogue no.: C8267
Batch: MKBG9425V
Expiry Date: September 2013
Route and Frequency
of Administration: orally, twice
Volume Administered: 10 mL/kg b.w.
Details on exposure:
orally
Frequency of treatment:
twice
Doses / concentrations
Remarks:
Doses / Concentrations:
1000, 500, 250
Basis:

No. of animals per sex per dose:
6 males and 6 females in the pre-experiments
50 males in the main experiment
9 males and 6 females for serum analytics
Control animals:
yes
Positive control(s):
Manufacturer: Acros Organics
Supplier: Fisher Scientific GmbH
Catalogue no.: 203960010
Charge: A0277203
Expiry Date: July 2013
Dissolved in: sterile water
Dosing: 40 mg/kg b.w.
Route and frequency
of administration: orally, once
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and 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 using a syringe. 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 (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
Evaluation criteria:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal at least 2000 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 2000 erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.

The study is considered valid if the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the negative control.
Statistics:
nonparametric Mann-Whitney test

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The animals treated in the pre-experiments received the test item CAT-Acid dissolved in corn oil twice orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested and expressed toxic reactions were shown in the table:

hours post
1. application hours post
2. application
1 2-4 6 24 1 2-4 6 24
1st Pre-experiment: 2 x 500 mg/kg b.w. males / females
reduction of spontaneous activity 2/2 2/2 0/0 0/0 0/0 0/0 0/0 0/0
ruffled fur 0/0 2/2 2/2 2/2 2/2 2/2 2/2 2/2
2nd Pre-experiment: 2 x 1000 mg/kg b.w. males / females
reduction of spontaneous activity 0/1 2/2 2/2 0/0 1/0 1/1 2/1 0/0
abdominal position 0/0 0/0 0/0 0/0 1/0 0/0 0/0 0/0
eyelid closure 0/1 0/2 1/2 0/0 0/0 1/1 1/1 0/0
ruffled fur 0/1 2/2 2/2 0/0 2/2 2/2 2/2 2/2
tumbling 0/0 0/0 0/0 0/0 0/2 0/1 0/0 0/0
hunchback 0/0 0/1 0/1 0/0 0/0 0/0 0/0 0/0
3rd Pre-experiment: 2 x 1500 mg/kg b.w. males / females
reduction of spontaneous activity 2/2 2/2 2/2 0/0 -/2 -/2 -/- -/-
abdominal position 2/2 2/2 2/2 0/0 -/2 -/1 -/- -/-
eyelid closure 0/0 1/1 2/2 0/0 -/1 -/2 -/- -/-
tumbling 2/2 2/2 2/2 0/0 -/2 -/0 -/- -/-
hunchback 0/0 1/1 1/2 0/0 -/0 -/1 -/- -/-
apathy 0/0 0/0 0/0 0/0 -/0 -/1 -/- -/-
ruffled fur 2/2 2/2 2/2 2/2 -/2 -/2 -/- -/-
death 0/0 0/0 0/0 2*/0 -/0 -/2** -/- -/-
*: both male animals were found death
**: both female animals were found moribund and were sacrificed
On the basis of these data 1000 mg/kg b.w. were estimated to be suitable as highest dose. No substantial sex specific differences were observed with regard to clinical signs. In agreement with the sponsor the main study was performed using males only.


RESULTS OF ANIMALS FOR BIOANALYTICS

The animals treated in the test groups for bioanalytics received the test item CAT-Acid dissolved in corn oil twice orally. The volume administered was 10 mL/kg b.w.. The following dose level was tested and expressed toxic reactions were shown in the table:

hours post
1. application hours post
2. application
1 2-4 6 24 1 2-4
2 x 1000 mg/kg b.w. males / females
reduction of spontaneous activity 0/4 0/0 0/1 0/0 6/6 3/3
ruffled fur 0/6 0/6 0/2 0/0 9/6 3/3
tumbling 0/0 0/0 0/0 0/0 4/4 2/3


RESULTS OF DEFINITIVE STUDY

In the main experiment for each test item dose groups 7 males received the test item CAT-Acid dissolved in corn oil twice orally. The volume administered was 10 mL/kg b.w.. The animals treated with the high, mid and low dose of the test item as well as the negative control (corn oil) did not express any toxic reactions.

Any other information on results incl. tables

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes
scoring 24 hours after second treatment with the test item.

Table1: vehicle

  16

m

corn oil

0

3

        1381

  17

m

 

 

2

        1329

  18

m

 

 

0

        1302

  19

m

 

 

4

        1235

  20

m

 

 

3

        1391

  21

m

 

 

3

        1365

  22

m

 

 

2

        1372

 

sum

17

        9375

 

mean

2.4

        1339

 

percent cells with micronuclei

0.121

 

Table2: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  23

m

CAT-Acid

250

0

        1348

  24

m

 

 

1

        1327

  25

m

 

 

2

        1339

  26

m

 

 

1

        1381

  27

m

 

 

1

        1464

  28

m

 

 

1

        1347

  29

m

 

 

0

        1376

 

sum

6

        9582

 

mean

0.9

        1369

 

percent cells with micronuclei

0.043

 


Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE/ total erythrocytes
scoring 24 hours after second treatment with the test item.

Table3: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  30

m

CAT-Acid

500

5

        1472

  31

m

 

 

2

        1419

  32

m

 

 

1

        1352

  33

m

 

 

1

        1336

  34

m

 

 

2

        1488

  35

m

 

 

1

        1379

  36

m

 

 

1

        1194

 

sum

13

        9640

 

mean

1.9

        1377

 

percent cells with micronuclei

0.093

 

Table4: test item

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  37

m

CAT-Acid

1000

3

        1277

  38

m

 

 

1

        1333

  39

m

 

 

1

        1230

  40

m

 

 

4

        1228

  41

m

 

 

3

        1222

  42

m

 

 

1

        1234

  43

m

 

 

2

        1285

 

sum

15

        8809

 

mean

2.1

        1258

 

percent cells with micronuclei

0.107

 

 Table5: positive control

animal no.

sex

test group

dose mg/kg b.w.

micronucleated cells per
2000 PCEs per animal

PCE per 2000 erythrocytes

  44

m

Cyclophosphamide

40

59

        1182

  45

m

 

 

48

        1241

  46

m

 

 

46

        1300

  47

m

 

 

25

        1501

  48

m

 

 

18

        1347

  49

m

 

 

50

        1308

  50

m

 

 

47

        1289

 

sum

293

        9168

 

mean

41.9

        1310

 

percent cells with micronuclei

2.093

 

Summary of Micronucleus Test Results

test group

dose mg/kg b.w.

sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythrocytes

vehicle

          0

    24

0.121

 0 -4

         1339

test item

      250

    24

0.043

 0 -2

         1369

test item

      500

    24

0.093

 1 -5

         1377

test item

    1000

    24

0.107

 1 -4

         1258

positive control

        40

    24

2.093

18 -59

         1310

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control
versus test group

Significance

p

 250 mg CAT-Acid/kg b.w.; 24 h

n.t.

-

 500 mg CAT-Acid/kg b.w.; 24 h

n.t.

-

1000 mg CAT-Acid/kg b.w.; 24 h

n.t.

-

   40 mg CPA/kg b.w.; 24 h

+

0.0003

      +    =    significant
      n.t. =    not tested

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The test item CAT-Acid was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally twice at an interval of 24 hwas 10 mL/kg b.w.. 48 h after the first administration of the test item (24 h after the last treatment) the bone marrow cells were collected for micronuclei analysis.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe 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 2000 erythrocytes.

As estimated by pre-experiments 1000 mg CAT-Acid, technical per kg b.w. twice orally 2 x 1000 mg/kg b.w. administered was suitable as highest treatment dose.

The bioanalysis for the pre-experiment (Non GLP) showed, that the test item could be detected in the plasma of the animals treated with the test item. The results of the test item levels in the plasma at the 1 h and 4 h post treatment sampling intervals were in the range of 5.87 – 42.3 and 5.55 – 32.4 µg/mL for the males and in the range of 26.4 – 98.2 and 12.7 – 20.7 µg/mL for the females, respectively. Thus, the bioavailability of the test item after an twice oral application could be confirmed and a main experiment was performed.

The following dose levels of the test item were investigated in the mutagenicity experiment: twice orally (2 x 250, 2 x 500, and 2 x 1000 mg/kg b.w.).

The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that CAT-Acid did not have any cytotoxic properties in the bone marrow.

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 CAT-Acid were below the value of the vehicle control group and within the historical control data.

Cyclophosphamide administered once orally (40 mg/kg b.w.) was used as positive control which showed a substantial and biologically relevant increase of induced micronucleus frequency.