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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 July 2014 (Study Plan completion) to 19 September 2014 (Quality Assurance statement)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Trilithium hexafluoroaluminate
EC Number:
237-509-4
EC Name:
Trilithium hexafluoroaluminate
Cas Number:
13821-20-0
Molecular formula:
AlF6.3Li
IUPAC Name:
trilithium(1+) hexafluoroalumanetriuide
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material : Lithium cryolite
- Molecular formula : Li3AlF6
- Molecular weight : 162
- Physical state: White powder
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: The animals were provided by Charles River, Sulzfeld, Germany.
- Age at study initiation: Young adult animals were selected (6-8 weeks old at the start of treatment).
- Number of animals : The total number of animals used in the dose range finding study was 8 and in the main study 33. In the micronucleus main study 5 male mice were treated per sampling time in each treatment group.
- Weight at study initiation: The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mean body weights were 36.2 ± 2.0 g and the range was 32 – 40 g.
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld 3-4 h prior to dosing until administration of Lithium cryolite
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height: 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15). A shelter (disposable paper corner home) and paper bedding (Enviro-dri) was provided as cage-enrichment.
- Diet : The animals had free access to pelleted rodent diet
- Water : The animals had free access to tap-water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
A controlled environment was maintained in the room with optimal conditions of approximately 10 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.7 - 22.5°C), a relative humidity of 40 - 70% (actual range: 38 - 64%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the minimum level for humidity (with max. 2%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.
- Concentration of test material in vehicle: no data
- Amount of vehicle : no data
- Type and concentration of dispersant aid (if powder): none
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Lithium cryolite was suspended in propylene glycol. Lithium cryolite concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Lithium cryolite concentrations were dosed within 4 hours after preparation.

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium cryolite. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. Feed was withheld 3-4 h prior to dosing until administration of Lithium cryolite.
The dosing volume was 5 ml/kg body weight.

Lithium cryolite concentrations were prepared on the day of administration.
Duration of treatment / exposure:
Range finding study: single dose, the observation period after dosing was one to 4 days. During this period mortality and physical condition were recorded at least once a day.

Main study : single dose. Three dose levels were used at the first sampling time. At the second sampling time only the highest dose was used. The first sampling time was 24 h after treatment and the second sampling time was 48 h after treatment.
Frequency of treatment:
Single dose.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
Dose range finding study : 1500 and 2000 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
Main study : 500, 1000 and 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Range finding study :
at 1500 mg/kg : 1 male and 1 female
at 2000 mg/kg: 3 males and 3 females

Main study:
Based on the results of the dose range finding test a full study with one sex was performed. Since there were no substantial differences in toxicity between sexes, only male animals were used in the main study.
At least five male mice were used per sampling time in each treatment group. The animals were dosed once.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0) dissolved in physiological saline dosed as a single oral intubation of 40 mg/kg body weight.
The route and frequency of administration of the positive control were the same as those of the test substance.

Examinations

Details of tissue and slide preparation:
ISOLATION OF BONE MARROW:
Bone marrow of the groups treated with Lithium cryolite was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were
sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.

PREPARATION OF BONE MARROW SMEARS
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol /ether and cleaned with a tissue. The slides were marked with the study identification number and the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

STAINING OF THE BONE MARROW SMEARS
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated coverslipper.

ANALYSIS OF THE BONE MARROW SMEARS FOR MICRONUCLEI
To prevent bias, all slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. In the group of the animals treated with the positive control substance only four animals were used for the scoring of micronucleated polychromatic erythrocytes.
Evaluation criteria:
Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals:
One male and one female animal were dosed with 1500 mg/kg bw. Within 1 hour after dosing both animals had a hunched posture.
In total three male and three female animals were dosed with 2000 mg/kg bw. No signs of toxicity were observed in one male and one female animal within 2 days after dosing. Within 2 hours after dosing 2 female and 2 male animals had a hunched posture. One male and female animal had recovered from the treatment within 21 hours after dosing and one female animal had recovered from the treatment within 47 hours. Within 71 hours after dosing one of the recovered female animals was lethargic, had a rough coat and a hunched posture. One male animal died within 47 hours after dosing.

RESULTS OF DEFINITIVE STUDY
- Doses: 500, 1500 and 2000 mg/kg bw
- Mortality and toxic signs:
The animals of the groups treated with 500 and 1000 mg /kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Three animals dosed with 2000 mg/kg bw had a hunched posture within 2 hours after dosing. Within 20 hours after dosing all animals were recovered or showed no reaction to treatment.
- Micronucleated polychromatic erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in Lithium cryolite treated groups were compared with the corresponding vehicle control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Lithium cryolite treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.
- Ratio polychromatic to normochromatic erythrocytes:
The animals of the groups, which were treated with Lithium cryolite showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Any other information on results incl. tables

Table 1: Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes

Group Treatment Dose (mg/kg bw) Sampling time (h) Number of micronucleated polychromatic erythrocytes (mean +/- SD)(1,3) Ratio polychromatic/ normochromatic erythrocytes (mean +/- SD)(1,4)
A Vehicle control 0 24 2.0 ± 1.0 0.94 ± 0.06 
B Lithium cryolite 2000 24 2.6 ± 1.1  0.83 ± 0.09
C Lithium cryolite 2000 48 2.4 ± 1.1 0.92 ± 0.04
D Lithium cryolite 1000 24 2.0 ± 0.7 0.90 ± 0.05 
E Lithium cryolite 500 24 2.6 ± 0.9  0.83 ± 0.13
F CP 40 48 19.5 ± 2.5(2,5) 0.59 ± 0.08(2)
Vehicle control =propylene glycol 
CP = Cyclophosphamide.
(1) Five animals per treatment group. 
(2) Four animals. 
(3) At least 2000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(4) The ratio was determined from at least the first 1000 erythrocytes counted.
(5) Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Table 2: INDIVIDUAL DATA

Individual data (males)

(group A       : oral intubation of the vehicle)

(group B & C: oral intubation of Lithium cryolite at 2000 mg/kg body weight)

(group D       : oral intubation of Lithium cryolite at 1000 mg/kg body weight)

(group E       : oral intubation of Lithium cryolite at 500 mg/kg body weight)

(group F        : oral intubation of cyclophosphamide at 40 mg/kg body weight)

 

 

 

 

Group

 

Animal number

 

Number of micronucleated polychromatic erythrocytes

Number of polychromatic erythrocytes

scored for micronuclei

 

Number of poly-chromatic erythrocytes(1)

 

Number of normo-chromatic erythrocytes(1)

 

Ratio polychromatic/

normochromatic

erythrocytes(1)

 

 

 

 

 

 

 

 

 

A

1

3

2013

498

503

0.99

 

A

2

3

2005

469

549

0.85

 

A

3

2

2009

516

558

0.92

 

A

4

1

2004

515

511

1.01

 

A

5

1

2021

510

555

0.92

 

 

 

 

 

 

 

 

 

B

6

4

2079

428

607

0.71

 

B

7

1

2052

459

559

0.82

 

B

8

3

2021

557

581

0.96

 

B

9

3

2001

459

547

0.84

 

B

10

2

2059

471

563

0.84

 

 

 

 

 

 

 

 

 

C

11

2

2001

474

541

0.88

 

C

12

2

2029

502

530

0.95

 

C

13

3

2009

492

511

0.96

 

C

14

4

2017

475

537

0.88

 

C

15

1

2013

506

546

0.93

 

 

 

 

 

 

 

 

 

D

16

3

2007

468

547

0.86

 

D

17

1

2093

459

547

0.84

 

D

18

2

2003

487

520

0.94

 

D

19

2

2017

519

561

0.93

 

D

20

2

2015

490

527

0.93

 

 

 

 

 

 

 

 

 

E

21

4

2043

507

542

0.94

 

E

22

3

2005

472

532

0.89

 

E

23

2

2019

492

559

0.88

 

E

24

2

2051

469

571

0.82

 

E

25

2

2035

383

633

0.61

 

 

 

 

 

 

 

 

 

F

26

23

2011

390

631

0.62

 

F

27

19

2009

407

598

0.68

 

F

28

19

2007

382

668

0.57

 

F

29

17

2001

332

677

0.49

 

F

   30(2)

 

 

 

 

 

 

 

 

 

 

 

 

 

(1) The ratio was determined from the first 1000 erythrocytes counted.

(2) Slide was not judged, hardly any cells present

Table 3 : statistics

Wilcoxon Rank Sum Test.
Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes; treatment/control comparison.
Group Treatment Dose Sex P-value Decision at 95%
    mg/kg bw   (one-sided) confidence level
F cyclophosphamide 40 males 0.01 significant

Validity criteria

A micronucleus test is considered acceptable if it meets the following criteria:

1) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range. => fulfilled, see table 1 2) The positive control substance induced a statistically significant (Wilcoxon Rank Sum, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes => fulfilled, see table 3 3) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range => fulfilled, 2 +/- 1 in the control, 1.1 +/-1.2 in the historical control. List of protocol deviations : In the positive control group, the slides of only four animals were used for the determination of the number of micronucleated polychromatic erythrocytes instead of five animals as specified in the protocol. Evaluation: The quality of the slides of one animal was poor. Hardly any cells were present. Since four analysable slides could be used for this positive control group and the incidence of micronucleated polychromatic erythrocytes was above the historical control data range (10-fold increase), this deviation had no effect on the results of the study. The study integrity was not adversely affected by the deviation. List of standard operating procedures deviations :There were no deviations from standard operating procedures that affected the integrity of the study.

Applicant's summary and conclusion

Conclusions:
Lithium cryolite is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a
dose of 2000 mg/kg under the experimental conditions described in this report.
Executive summary:

Lithium cryolite was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. The test was performed according to the OECD Testing Guideline 474 and under the GLP.

In the dose range finding study 1 male and 1 female were dosed once via oral gavage with 1500 mg Lithium cryolite per kg body weight. Since the animals showed only a hunched posture directly after treatment, an additional 3 males and 3 females were dosed once via oral gavage with 2000 mg Lithium cryolite per kg body weight. Within 2 hours after dosing 2 female and 2 male animals had a hunched posture. One male and female animal had recovered from the treatment within 21 hours after dosing and one female animal had recovered from the treatment within 47 hours. Within 71 hours after dosing one of the recovered female animals was lethargic, had a rough coat and a hunched posture. One male animal died within 47 hours after dosing.

In the main study male animals were dosed once via oral gavage with vehicle or with 2000, 1000 and 500 mg/kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals.

Clinical signs of toxicity were limited to the high dose group and included a hunched posture (3 animals).

Bone marrow of the groups treated with Lithium cryolite was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with Lithium cryolite compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with Lithium cryolite showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that Lithium cryolite is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions described in this report.