Reaction mass of Potassium sodium 3-{[4-({3-(substitutedamino)-4-[(2-sulfonato-4-{[2-(sulfonatooxy)ethyl]sulfonyl}phenyl)diazenyl]phenyl}amino)-6-chloro-heteromonocyl-2-yl]amino}-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (1:3:1) and Potassium sodium 3-[(4-{[3-(subsitutedamino)-4-{[4-(ethenylsulfonyl)-2-sulfonatophenyl]diazenyl}phenyl]amino}-6-chloro-heteromonocy-2-yl)amino]-2-substituted-5-{[2-(sulfonatooxy)ethyl]sulfonyl}benzenesulfonate (3:9:3)

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:

2014-03-20 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 material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 40863/A TE
Batch: BOP 02-13 (BS-ROE1670)
Purity: 82.1 %, dose calculation was not adjusted to purity
Physical Appearance: Brownish powder, solid at 20°C
Retest Date: 05 June 2018
Storage Conditions: At room temperature
Stability in Solvent: Not indicated by the Sponsor

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): 2 males and 2 females
Number of animals (main study): 28 males
Age: 8 - 9 weeks (beginning of treatment)
Body weight: mean value 34.7 g (SD  1.7 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: 20 March 2014 To: 09 April 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Water: sterile
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:
2000 mg/kg b.w.
Basis:

No. of animals per sex per dose:

2 males and 2 females used in the pre-experiment
28 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. 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.

Statistics:

nonparametric Mann-Whitney test; p <0.05

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The animals treated in the pre-experiment received the test item FAT 40863/A TE dissolved in sterile water once orally. The volume administered was 10 mL/kg b.w.. No signs of toxicity were observed. The faeces of the animals were red to brown coloured after treatment with the test item.
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.
In agreement with the Sponsor, a limit test with 2000 mg/kg was performed.

RESULTS OF DEFINITIVE STUDY
In the main experiment for each test item dose group 7 males received the test item FAT 40863/A TE dissolved in sterile water once orally. The volume administered was 10 mL/kg b.w.. No clinical symptoms were observed in the test item treated animals. The faeces of the animals were red to brown coloured after treatment with the test item.
The animals treated with the vehicle control (sterile water) 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	0.071	0 - 4	1180
test item	2000	24	0.107	0 - 5	1242
positive control	40	24	1.971	23 - 73	1174
test item	2000	48	0.179	1 - 7	1318

  

 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
2000 mgFAT 40863/A TE/kg b.w.; 24 h	-	0.2197
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mgFAT 40863/A TE/kg b.w.; 48 h	+	0.0373

                  -       =not significant
                 +      =significant;

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	sterile water	0	0	1277
2	m			4	1162
3	m			0	1233
4	m			4	1100
5	m			0	1120
6	m			1	1144
7	m			1	1225
	sum			10	8261
	mean			1.4	1180
	percent cells with micronuclei			0.071

 

Table2: 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
8	m	FAT 40863/ATE	2000	1	1341
9	m			1	1243
10	m			5	1189
11	m			0	1218
12	m			4	1295
13	m			2	1139
14	m			2	1266
	sum			15	8691
	mean			2.1	1242
	percent cells with micronuclei			0.107

 

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
15		m		CPA		40		23		1359
16		m						46		1074
17		m						73		1158
18		m						35		1179
19		m						34		1110
20		m						30		1214
21		m						35		1126
		sum				276		8220
		mean				39.4		1174
		percent cells with micronuclei				1.971

 

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
22	m	FAT 40863/ATE	2000	3	1328
23	m			4	1271
24	m			4	1336
25	m			4	1322
26	m			7	1363
27	m			2	1357
28	m			1	1247
	sum			25	9224
	mean			3.6	1318
	percent cells with micronuclei			0.179

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 40863/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 40863/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 dissolved in sterile water, 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 red to brown coloured after treatment with the test item in the pre-experiment and main experiment.

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

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FAT 40863/A TE did not have any cytotoxic properties 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 for the high dose level 24 hour after administration of the test item. A statistically significant increase was observed on animals treated with the high dose level 48 hours of treatment. However, all values observed in the test item treated dose groups at any preparation interval were very well within the laboratory’s historical vehicle control data. Moreover, except for two animals (animal 26: 7 micronucleated cells/ 2000 PCE, animal 10: 5 micronucleated cells/ 2000 PCE) the frequency of micronucleated cells in the single animals was not higher than that of the vehicle control animals (maximum of 4 micronucleated cells/2000 PCE). Thus, the observed increase was not considered as biologically relevant.

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 40863/A TEdid not induce micronucleias determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, FAT 40863/A TE is considered to be non-mutagenic in this micronucleus assay.