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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
A bacterial reverse mutation test, in vitro chromosome aberration test in Human lymphocytes test, a mouse lymphoma test and an in vivo micronucleus test performed according to OECD guidelines and GLP principles were available. FAT 40863/A TE was found to be not mutagenic in the bacterial reverse mutation assay and in the mouse lymphoma assay in the absence and presence of S9-mix. FAT 40863/A TE was not found to be clastogenic in the chromosome aberration test with and without metabolic activation. In an in vivo micronucleus test FAT 40863/A TE was no mutagenic.
Link to relevant study records
Reference
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
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals 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
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
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.
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
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.

 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

 



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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Several mutagenicity studies have been conducted in in-vitro test systems which all turned out to be negative with and without metabolic activation, indicating clearly a non mutagenic potential in-vitro.

One in-vivo mutagenicity assays was performed: Micronucleus Assay in Bone Marrow Cells of the Mouse and FAT 40863/A TE has no significant mutagenic potential in-vivo.

Altogether, it was judged that FAT 40863/A TE substance is non-mutagenic in vitroand not mutagenic in in-vivo experiment.


Justification for selection of genetic toxicity endpoint
A reliable bacterial reverse mutation test, in vitro chromosome aberration in Human lymphocyte test, mouse lymphoma test and in vivo micronucleus test were test are available and were all performed according to OECD/EC guidelines.
All these four studies are negative and all of them are taken into consideration

Justification for classification or non-classification

Based on the above mentioned results the substance does not need to be classified according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).