Reactions mass of N-{2-[(4-bromo-6-substituted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-methoxyalkyl)amino]phenyl}acetamide) and N-{2-[(4-bromo-6-substiuted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-hydroxy-3-phenoxypropyl)amino]phenyl}acetamide

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:

key study

Study period:

2014-03-17 to 2014-04-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Test system: Mice, NMRI
Rationale: Recognised as the recommended test system.
Source: Charles River Laboratories
Research Models and Services Germany GmbH
Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of animals (pre-test): 4 males and 4 females (2 per sex for each pre-test)
Number of animals (main study): 42 males
Age: 8 - 10 weeks (beginning of treatment)
Body weight: mean value 37.6 g (SD +/- 2.2 g)

ENVIRONMENTAL CONDITIONS
Housing: single
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity approx. 27.1 - 65%
artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: 17 March 2014 To: 09 April 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

DMSO / PEG 400 (3/7): 30% Dimethyl sulphoxide / 70 % Polyethylenglycol
Route and frequency of administration: orally once
Volume administered: 10 mL/kg b.w.

Details on exposure:

orally

Frequency of treatment:

once

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 100, and 2000 mg/kg b.w.
Basis:

No. of animals per sex per dose:

4 males and 4 females (2 per sex for each pre-test)
42 males used in the main experiment

Control animals:

yes

Positive control(s):

Cyclophosphamide: purity: >98%; dissolved in sterile water
Route and frequency of administration: orally once
Concentration: 40 mg/kg b.w.
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 FBS 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/Giemsa. Cover slips were mounted with EUKITT. 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. At least per animal 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 as the following criteria are met:
- at least 5 animals per group could be evaluated.
- PCE to erythrocyte ratio was not less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.
- the rate of micronucleated PCEs in the negative control falls within the historical laboratory control data range

Statistics:

nonparametric Mann-Whitney test; p<0.05

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle 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 FAT 41044/A TE suspended in DMSO / PEG 400 (3/7) once orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested and expressed clinical symptoms were shown in the table:
hours post-treatment
Clinical Symptoms 0-1 2-4 5-6 24 30 48

1st Pre-experiment: 1000 mg/kg b.w.; males / females
ruffled fur 1/0 1/0 0/0 0/0 0/0 0/0

2nd Pre-experiment: 2000 mg/kg b.w. ; males / females
No toxic reactions were observed.

The faeces of the animals were blue coloured after treatment with the test item in the first and second pre-experiment.

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable. No substantial differences between sexes in toxicity were observed, so that only male animals were used in the main experiment.


RESULTS OF DEFINITIVE STUDY
In the main experiment for each test item dose group 7 males received the test item FAT 41044/A TE suspended in DMSO / PEG 400 (3/7) once orally. The volume administered was 10 mL/kg b.w.. The clinical symptoms observed following treatment are shown in the following table for each dose group, which indicates the number of animals with findings.
hours post-treatment (males)
Clinical symptoms 1 2-4 6 24 48*

High dose: 2000 mg/kg b.w.
No toxic reactions were observed.

Medium dose: 1000 mg/kg b.w.*
ruffled fur 1 1 0 0 -

Low dose: 500 mg/kg b.w.*
No toxic reactions were observed.

*: data only from 7 male animals
-: no observation made

The faeces of the animals were blue coloured after treatment with the test item in the main experiment for the mid and high dose group.
The animals treated with the vehicle control (DMSO / PEG 400 (3/7)) did not express any clinical symptoms.

Any other information on results incl. tables

 Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
vehicle control	0	24	0.086	0 - 4	1199
test item	500	24	0.064	0 - 4	1261
test item	1000	24	0.079	0 - 3	1161
test item	2000	24	0.057	0 - 4	1235
positive control	40	24	1.664	16 - 54	1163
test item	2000	48	0.057	0 - 2	1202

  

Biometry

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

 

Negative control versus test group	Significance	p
500 mgFAT 41044/A TE/kg b.w.; 24 h	n.t.	-
1000 mgFAT 41044/A TE/kg b.w.; 24 h	n.t.	-
2000 mgFAT 41044/A TE/kg b.w.; 24 h	n.t.	-
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mgFAT 41044/A TE/kg b.w.; 48 h	n.t.	-

                  -       =not significant
                 +      =significant;
                 n.t.   =not tested, as the mean micronucleus frequency was not above the vehicle
                           control value

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 erythrocytes scoring 24 hours after treatment.

Table1: Vehicle Control

 

animal no.	sex	test group	dose mg/kg b.w.	micronucleated cells per 2000 PCEs per animal	PCE per 2000 erythrocytes
1	m	DMSO/PEG 400 (3/7)	0	0	1229
2	m			0	1252
3	m			4	1151
4	m			3	1167
5	m			0	1310
6	m			2	1161
7	m			3	1121
	sum			12	8391
	mean			1.7	1199
	percent cells with micronuclei			0.086

 

Table2: Test Item –Low Dose Group

 

animal no.	sex	test group	dose mg/kg b.w.	micronucleated cells per 2000 PCEs per animal	PCE per 2000 erythrocytes
8	m	FAT 41044/A TE	500	0	1243
9	m			0	1315
10	m			0	1331
11	m			2	1220
12	m			0	1155
13	m			3	1331
14	m			4	1233
	sum			9	8828
	mean			1.3	1261
	percent cells with micronuclei			0.064

 

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 erythrocytes scoring 24 hours after treatment.

Table3: Test Item – Medium Dose Group

animal no.		sex		test group		dose mg/kg b.w.		micronucleated cells per 2000 PCEs per animal		PCE per 2000 erythrocytes
15		m		FAT 41044/A TE		1000		1		1175
16		m						1		1217
17		m						0		1132
18		m						0		1096
19		m						3		1091
20		m						3		1161
21		m						3		1255
		sum				11		8127
		mean				1.6		1161
		percent cells with micronuclei				0.079

 

Table4: Test Item – High Dose Group

animal no.		sex		test group		dose mg/kg b.w.		micronucleated cells per 2000 PCEs per animal		PCE per 2000 erythrocytes
22		m		FAT 41044/A TE		2000		4		1082
23		m						1		1298
24		m						1		1241
25		m						0		1266
26		m						2		1247
27		m						0		1258
28		m						0		1253
		sum				8		8645
		mean				1.1		1235
		percent cells with micronuclei				0.057

 

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 erythrocytes scoring 24 hours after treatment.

Table 3: Positive Control Group

 

animal no.		sex		test group		dose mg/kg b.w.		micronucleated cells per 2000 PCEs per animal		PCE per 2000 erythrocytes
29		m		CPA		40		26		1139
30		m						54		1114
31*		m						28		1006
32		m						34		1127
33		m						16		1282
34		m						30		1272
35		m						45		1143
		sum				233		8143
		mean				33.3		1163
		percent cells with micronuclei				1.664

*Sample size was raised to 6000 PCE due to inconsistent data

 

Micronuclei in polychromatic erythrocytes (PCE) and relationship PCE per 2000 erythrocytes scoring 48 hours after treatment.

Table 4: Test Item – High Dose Group

 

animal no.	sex	test group	dose mg/kg b.w.	micronucleated cells per 2000 PCEs per animal	PCE per 2000 erythrocytes
36	m	FAT 41044/A TE	2000	1	1102
37	m			2	1197
38	m			2	1248
39	m			1	1299
40	m			0	1228
41	m			1	1149
42	m			1	1192
	sum			8	8415
	mean			1.1	1202
	percent cells with micronuclei			0.057

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 FAT 41044/A TE did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

The test item FAT 41044/A TE 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 suspended in DMSO / PEG 400 (3/7), which was also used as vehicle control. The volume administered orally was 10mL/kg b.w. The volume of the positive control administered was 10 mL/kg. The faeces of the animals were blue coloured after treatment with the test item in the pre-experiment and in the main experiment for the mid and high dose group.

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. At least per animal 2000 polychromatic erythrocytes (PCEs) 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 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

The maximum guideline-recommended dose of 2000 mg/kg b.w. was investigated at preparation intervals of 24 and 48 h, respectively, based on two pre-experiments.

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 FAT 41044/A TE did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle control 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. In fact the mean values of micronuclei observed after treatment with FAT 41044/A TE were below to the value of the vehicle control group.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test item FAT 41044/A TEdid not induce micronucleias determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, FAT 41044/A TE is considered to be non-mutagenic in this micronucleus assay.