Cyclohexanecarboxylic acid, 1-(2-ethylbutyl)-

Genetic toxicity: in vivo

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

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

Test material information

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.