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:
key study
Study period:
Study was conducted between 28th July and 17th October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: FAT 20341/A TE
Physical State / Appearance: Black powder
Purity: 80.1%
Batch Number: BOP 02-12 (Navy PLK 241, BS)
Expiry Date: 21 November 2017
Storage Conditions: Room temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
Sufficient albino Hsd: ICR (CD-1®) strain mice were obtained from Harlan Laboratories UK Ltd., Oxon, UK. At the start of the main test the mice weighed 20.9 to 28.6 g and were approximately six to ten weeks old. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood flake bedding. Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.

The temperature and relative humidity were set to achieve limits of 19 to 25ºC and 30 to 70%, respectively. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vehicle control:
Information as provided by the supplier. The PBS for the main test was prepared by formulating as a 1x solution in distilled water (Laboratoire Aguettant lot: 3010598)

Identification: DPBS (10x) (-) CaCl2 (-) MgCl2
Batch Number: 1273659
Harlan Serial Number: V-5999
Expiry Date: 01 January 2015
Storage Conditions: Room temperature
Purity: Not supplied
Details on exposure:
Range finding study:
All animals were dosed once only at the appropriate dose level by either gavage using a metal cannula attached to a graduated syringe or ip injection using a hyperdermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of dosing.

Definitive study:
Groups, each of seven mice, were dosed once only via the ip route with the test item at 50, 25 or 12.5 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 50 mg/kg was killed after 48 hours. In addition, two further groups of mice were included in the study; one group (seven mice) was dosed via the ip route with the vehicle alone (PBS) and a second group (five mice) was dosed orally with cyclophosphamide. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test. The vehicle controls were killed 24 hours following dosing and positive control group animals were killed 24 hours following dosing.
Duration of treatment / exposure:
Range finding study:
The animals were dosed at the following concentrations 50, 200, 1000 and 2000 mg/kg. Animals were observed (where applicable) approximately 0.5, 1, 2, and 4 hours after dosing and subsequently once daily for two days. Any deaths and evidence of overt toxicity were recorded at each observation.

Definitive study:
24 hours in the following dose groups 0, 12.5, 25 and 50 mg/kg.
48 hours in the 50 mg/kg dose group.
Frequency of treatment:
Groups, each of seven mice, were dosed once only via the ip route with the test item at 50, 25 or 12.5 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 50 mg/kg was killed after 48 hours. In addition, two further groups of mice were included in the study; one group (seven mice) was dosed via the ip route with the vehicle alone (PBS) and a second group (five mice) was dosed orally with cyclophosphamide.
Post exposure period:
All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable and immediately prior to termination.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 12.5, 25 and 50 mg/kg.
Basis:
other: Dosed once only via the ip route.
No. of animals per sex per dose:
Preliminary test:
1 male and 1 female were dosed at the following concentrations: 50, 200, 1000 and 2000 mg/kg.
2 males were dosed at the following concentration: 50 mg/kg.

Mouse Micronucleus Test:
7 males were used per dose group (12.5 mg/kg, 25 mg/kg and 50 mg/kg). For the vehicle control, a smaller group of 5 males were used for the positive control.
Control animals:
yes
Positive control(s):
Positive control:
Identification: Cyclophosphamide
Acros Organics Lot Number: A0302605
Harlan Serial Number: R-5819
Purity: 97%
Expiry date: 06 January 2016
Storage Conditions: Approximately 4 ◦C

Examinations

Tissues and cell types examined:
Slide Evaluation:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.

The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Details of tissue and slide preparation:
Slide Preparation:
Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

Evaluation criteria:
Interpretation of results:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to the vehicle control group.

If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.

A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.



Statistics:
All data were statistically analyzed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analyzed following a √(x + 1) transformation using Student's t-test (two tailed).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The testes of the mice were grey in colour, hunched posture + ptosis were seen at 50 and 25 mg/kg groups. In the 24 hour exposure group at 12.5mg/kg the clinical signs of hunched posture and ptosis were seen + considered to be due to systemic exposure.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Range-finding Toxicity Test

The mortality data are summarized as follows:

 

Dose Level

(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

2000

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

1000

Male

1

ip

1e

0

0

2/2

Female

1

1e

0

0

200

Male

1

ip

0

1e

0

1/2

Female

1

0

0

0

50

Male

1

ip

0

0

0

0/2

Female

1

0

0

0

50

Male

2

ip

0

0

0

0/2

e= Animals killed in extremis

 

No clinical signs of toxicity were observed in animals dosed with the test item at 2000 mg/kg via the oral route. However the faeces was stained black in colour. Therefore the route of exposure was changed to ip to maximise the exposure. In animals dosed with the test item at 1000 mg/kg via the ip route, the following clinical signs were observed: prostration, decreased respiration, labored respiration, ptosis and the extremities were stained black in colour. Both animals were killed In extremis due to the severity of the clinical signs. In animals dosed with the test item at 200 mg/kg via the ip route, the following clinical signs were observed: hunched posture, ptosis, ataxia, lethargy, tiptoe gait, splayed gait, laboured respiration, hypothermia, faeces stained black in colour and extremities stained black in colour. One of the animals was killed in extremis due to the severity of the clinical signs. In animals dosed with the test item at 50 mg/kg via the ip route, the following clinical signs were observed: hunched posture, ptosis, ataxia and splayed gait. 50 mg/kg was considered to be the maximum tolerated dose level. There was no marked difference in toxicity between sexes therefore confirmatory male animals were dosed with the test item at 50 mg/kg and the clinical signs of hunched posture, ptosis and splayed gait were observed.

 

The test item showed no marked difference in its toxicity to male or female mice via ip administration, it was therefore considered to be acceptable to ip dose males only for the main test. With evidence of acceptable toxicity at the maximum tolerated dose (MTD) of the test item, 50 mg/kg was selected for use in the main test with 25 and 12.5 mg/kg as the lower dose levels.

 Micronucleus Test:

Mortality Data and Clinical Observations:

There were no premature deaths seen in any of the dose groups. The following clinical signs were observed at 50 and 25 mg/kg in both the 24 and 48-hour dose groups (where applicable): hunched posture, and ptosis. In addition the testes of the mice appeared grey/blue in colour. In the 24 hour exposure group at 12.5mg kg the clinical signs of hunched posture and ptosis were observed. These clinical signs were considered to be indicative of systemic exposure to the test item.

 

Evaluation of Bone Marrow Slides:

A summary of the results of the micronucleus test is given in Table 1*. Individual and group mean data are presented in Tables 2* to 7*.

 

There were no statistically significant decreases in the PCE/NCE ratio in any test item exposure group. 

 

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item dose groups when compared to the vehicle control group.

 

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

* Table 1 can be found below, tables 2 to 7 can eb found in attachment 1 which can be found within the attachments section. Table 1     Micronucleus Test – Summary of Group Mean Data

TREATMENT GROUP

NUMBER OF PCE WITH MICRONUCLEI PER 2000 PCE

PCE/NCE RATIO

GROUP MEAN

SD

GROUP MEAN

SD

1.  Vehicle Control

2.1

2.4

1.15

0.28

      24-hour sampling time

2.   Positive Control

56.6***

13.7

1.21

0.39

      24-hour sampling time

3.   FAT 20341/A TE

1.4

1.1

0.83

0.15

      50 mg/kg

      48-hour sampling time

4.   FAT 20341/A TE

1.1

1.2

0.83

0.32

      50 mg/kg

      24-hour sampling time

5.   FAT 20341/A TE

1.4

1.1

1.53

0.69

      25 mg/kg

      24-hour sampling time

6.   FAT 20341/A TE

2.3

1.4

1.89

1.23

      12.5 mg/kg

      24-hour sampling time


PCE      =Polychromatic erythrocytes

NCE     =Normochromatic erythrocytes

SD       =Standard deviation

***      = P < 0.001

 

 

 


 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item, FAT 20341/A TE, was considered to be non-genotoxic under the conditions of the test.
Executive summary:

Introduction:

The study was perford to assess the potential of the test item to produce damage to chromosos or aneuploidy when administered to mice. Thethod was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

Methods:

A range-finding test was performed to find suitable dose levels of the test item, route of administration and to investigate whether there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the ip (intraperitoneal) route in groups of seven mice (males) at the maximum tolerated dose of 50 mg/kg, with 25 and 12.5 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

 

 Additional groups of mice were given a single ip dose of PBS (7 mice), or dosed orally with cyclophosphamide, (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 hours later, and positive control animals were killed after 24 hours.

 

Results:

There were no premature deaths seen in any of the dose groups. The following clinical signs were observed at 50 and 25 mg/kg in both the 24 and 48-hour dose groups (where applicable): hunched posture and ptosis. In addition the testes had grey/blue coloration. In the 12.5 mg/kg dose group, the clinical signs of hunched posture and ptosis were observed.

 

No statistically significant decreases in the PCE/NCE ratio were observed in any test item dose groups when compared to the vehicle control group.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

 

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

Conclusion:

The test item, FAT20341/A TE, was considered to be non-genotoxic under the conditions of the test.