Lithium phosphorodifluoridate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Due to the observed mortality during the main experiment, an additional two replacement animals were used in the 100 mg/kg body weight dose group (24-hour sampling time point) to examine the required number of animals.

Test guideline 2

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:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

lithium(1+) (difluorophosphoryl)oxidanide

EC Number:

643-080-8

Cas Number:

24389-25-1

Molecular formula:

LiPO2F2

IUPAC Name:

lithium(1+) (difluorophosphoryl)oxidanide

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 8252153

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

RjHan

Details on species / strain selection:

The NMRI mouse is one of the standard animals used internationally in this type of mutagenicity testing

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier, Route des Chènes Secs B.P. 4105, 53940 LE GENEST-ST-ISLE, France
- Age at study initiation: approx. 7 weeks at treatment
- Weight at study initiation: 34.7 – 38.0 g (males, preliminary experiment), 28.2 – 31.8 g (females, preliminary experiment), 33.3 – 37.9 g (males, main test)
- Assigned to test groups randomly: yes, based on body weights SPSS/PC+ software was used in order to verify homogeneity/variation among/within groups.
- Fasting period before study:
- Housing: Group caging (5 animals/cage or 2 animals/cage) to allow social interaction, and with deep wood sawdust bedding, to allow digging and other normal rodent activities. Cage type: II. type polypropylene/polycarbonate
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for mice and rats breeding and maintenance" (Batch number: 523 7816, Expiry date: January 2013; and Batch Number: 445 8440; Expiry date: May 2013) produced by ssniff Spezialdiäten GmbH (D-59494, Soest, Germany)
- Water (e.g. ad libitum): tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.5°C
- Humidity (%): 31 - 70%
- Air changes (per hr): 15 - 20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12h dark, 12h light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Name: Distilled water
Supplier: TEVA
Batch number: 3450611
Expiry date: 30 June 2014
Storage conditions: Room temperature

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The necessary amount of the test item was weighed into a calibrated volumetric flask; the appropriate amount of vehicle was added and stirred to obtain homogenous formulations. The concentrations of the test item formulations were chosen to assure the same dosing volumes in mice for all dose levels (10 mL/kg bw). The test item was used for treatment in the main test at concentrations of 10, 5 and 2.5 mg/mL. The formulations were prepared immediately before the treatment

PRELIMINARY TOXICITY TESTING
Two preliminary toxicity tests were performed to identify the appropriate maximum dose level for the main test. The preliminary toxicity tests also determined whether there were large differences in toxicity between the sexes. Groups of two male and female mice were treated on one occasion by oral gavage at the dose levels of 300, 200, 100 and 50 mg/kg body weight (Preliminary Experiment I). Based on the observed mortality in the 300 and 200 mg/kg body weight dose groups, additional doses of 150 and 125 mg/kg body weight were also examined in an additional experiment (Preliminary Experiment II.)

MAIN TEST
Based on the results of the preliminary toxicity tests, 100 mg/kg body weight was selected for the highest dose level of the test item in the main micronucleus test. Two lower dose levels separated by a factor of two (50 and 25 mg/kg body weight) were also included in the main test. The dose levels were expressed in terms of the test item as received.

Duration of treatment / exposure:

single dose, oral gavage

Frequency of treatment:

once

Post exposure period:

48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Negative control (vehicle)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Test item (dose conc: 2.5 mg/mL)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Test item (dose conc.: 5 mg/mL)

Doses / concentrations 4

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Test item (Dose conc.: 10 mg/mL)

No. of animals per sex per dose:

Negative control (vehicle) = 10 males
25 mg/kg bw/day = 5 males
50 mg/kg bw/day = 10 males
100 mg/kg bw/day = 10 + 7 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclphosphamide (Dose conc. 6 mg/mL)

Examinations

Tissues and cell types examined:

bone marrow
polychromatic erthrocytes (PCEs)
normochromatic erythrocytes (NCEs)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
negative control (vehicle) = sampling 24h and 48h after treatment
25 mg/kg bw/day = sampling 24h after treatment
50 mg/kg bw/day = sampling 24h after treatment
100 mg/kg bw/day = sampling 24h and 48h after treatment

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time in the main test, mice were euthanized by asphyxiation with ascending doses of carbon dioxide. Deep anaesthesia was confirmed before cervical dislocation or transection of the major cervical blood vessels before confirming death and discarding carcasses. Bone marrow was obtained from two exposed femurs of mice* immediately after sacrifice.

The bone marrow was flushed out of each pair of femurs with foetal bovine serum (5 mL) using a syringe and needle into a sterile centrifuge tube. After mixing, the cell suspension was concentrated by a gentle centrifugation and the supernatant was discarded. Smears of the cell pellet were made on standard microscope slides (2 slides / animal). Slides were air-dried at room temperature for approximately 24 hours.

Dried slides were fixed in methanol for a minimum of 5 minutes and allowed to air-dry. Two slides per animal were stained with 10 % Giemsa solution for 20 minutes then thoroughly rinsed with distilled water, and then air-dried at room temperature for at least 12 hours. After staining, coverslips were mounted on them.

METHOD OF ANALYSIS:
Prior to microscope analysis, the stained slides were given unique code numbers by a person who was not involved in the analysis. The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the incidence of the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. Fewer than 2000 PCEs may be counted in the event of a clear positive effect.
The proportion of immature among total (immature + mature) erythrocytes was be determined for each animal by counting a total of at least 1000 cells (immature erythrocytes, PCEs plus mature normochromatic erythrocytes, NCEs), in which the number of micronuclei were recorded in both types of erythrocytes.

Evaluation criteria:

Criteria for a positive response:
The test item is considered to have shown genotoxic activity if statistically significant increases in the frequency of micronucleated polychromatic erythrocytes are observed in treated animals compared to the corresponding negative controls, and the increases are dose-related. Historical control data are taken into consideration when evaluating the biological significance of small increases.

Criteria for a negative response:
The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed above the concurrent and historical control values.

Equivocal response:
Results which do not meet the criteria for a positive or negative response are considered to be equivocal. Further investigations or scoring of additional cells may be necessary in case of an equivocal result.

Statistics:

Kruskal Wallis test

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Based on the available information, the test item was soluble in Distilled water. As this vehicle was compatible to the test system, it was selected as vehicle for the study. Groups of two male and female mice were treated on one occasion by oral gavage at the dose levels of 300, 200, 100 and 50 mg/kg body weight (Preliminary Experiment I). The treatment volume was 10 mL/kg body weight. Animals were examined regularly for toxic signs and mortalities. Based on the observed mortality in the 300 and 200 mg/kg body weight dose groups, additional doses of 150 and 125 mg/kg body weight were also examined in an additional experiment (Preliminary Experiment II.). All the surviving mice were euthanized 48 hours after treatment.

There were some individual variability in the time of the appearance or severity of the symptoms, but both male and female animals died or were considered moribund in the 300, 200, 150 and 125 mg/kg body weight dose groups. Decreased activity was detected for all animals of the 100 mg/kg body weight dose group, in some animals hunched back, prone position and/or piloerection was also observed.

All animals were free of clinical signs in the 50 mg/kg body weight dose group. No treatment related effect on body weight was observed in the 100 and 50 mg/ kg body weight group.

Based on the results of the preliminary toxicity test, dose levels of 100, 50 and 25 mg/kg body weight were selected for the micronucleus test. As the toxicity of the test item was similar in both sexes in the preliminary toxicity tests, the main experiment was performed using male mice only.

RESULTS OF DEFINITIVE STUDY
Marked body weight loss (>10%) was detected for 1 of 7 surviving animals in the high dose group at the 24-hour sampling time point and for 2 of 6 surviving animals in the high dose group at the 48-hour sampling time point in the main test. No effect on the body weight was observed in the mid and low dose groups, or in the negative or positive control groups.

Mortality was observed in the high dose group (100 mg/kg body weight) in the main test: 4 animals were found dead from the 17 treated animals at different time points. Signs of systemic toxicity (decreased activity, hunched back, prone position or intermittent tremors) and piloerection were also observed for some animals in this dose group. The animals in the negative (vehicle) control, positive control and low and mid dose groups were symptom-free during the whole observation period (with the exception of one animal in the mid dose group (50 mg/kg body weight) where piloerection was observed).

Two thousand polychromatic erythrocytes (PCEs) were scored per animal to assess the micronucleated cells. The frequency of micronucleated cells was expressed as percent of micronucleated cells based on the first 2000 PCEs counted in the optic field. The proportion of immature among total erythrocytes was determined for each animal by also counting mature erythrocytes (NCEs) until a total of 1000 PCEs+NCEs had been observed.

The group treated for 24 hours with 100 mg/kg bw/day, which gave the highest number of micronuclei, was compared with the corresponding negative (vehicle) control group using the Kruskal Wallis test. This gave a value of H = 0.561 which is non-significant, giving a negative response. All other groups showed the same or lower numbers of micronuclei than the corresponding negative (vehicle) control group.

The positive and negative control results were also compared, and gave a value of H = 6.991 (p<0.01). The positive control treatment therefore caused a very substantial increase, demonstrating the sensitivity of the test system. The positive and negative control data were considered to give adequate data to confirm the validity of the study.

The frequency of micronucleated polychromatic erythrocytes of the negative (vehicle) control group was within the range of historical laboratory control data; the positive control item produced a biologically and statistically relevant increase in the number of micronucleated polychromatic erythrocytes; each treated and control group included at least 5 analysable animals; therefore, the test was considered to be valid.

Any other information on results incl. tables

Table 1: Micronucleus data (24 hours after dosing)

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Negative control (vehicle)	7	0	558
	22	2	601
	24	2	535
	47	2	397
	42	3	400
Mean		1.8	498.2
S.D		1.10	94.05

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Test item 25 mg/kg bw	9	1	333
	13	1	397
	34	3	603
	36	2	490
	39	2	569
Mean		1.8	478.4
S.D		0.84	113.62

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Test item 50 mg/kg bw	6	1	282
	12	2	505
	16	0	594
	20	2	592
	33	0	505
Mean		1	495.6
S.D		1.00	127.26

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Test item 100 mg/kg bw	3	3	615
	8	5	550
	23	0	536
	28	1	592
	47	5	608
Mean		2.8	580.2
S.D		2.28	35.32

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Positive control (cyclophosphamide)	11	36	427
	15	56	514
	29	49	425
	46	51	463
	49	56	474
Mean		49.6	460.6
S.D		8.20	36.86

MNPCE: Number of Micronucleated Polychromatic Erythrocytes referring to counts of 2000 PCE.

PCE: Polychromatic Erythrocyte

NCE: Normochromatic Erythrocyte

Table 2: Micronucleus data (48 hours after dosing)

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Negative control (vehicle)	14	3	486
	18	3	447
	40	3	435
	43	2	458
	45	2	384
Mean		2.6	442.0
S.D		0.55	37.52

Treatment	Animal number	MNPCE/2000 PCE	PCE/1000 PCE + NCE
Test item 100 mg/kg bw	2	0	276
	30	1	523
	32	3	429
	35	3	406
	41	0	520
Mean		1.4	430.8
S.D		1.52	101.29

Applicant's summary and conclusion

Conclusions:

In conclusion, no induction of micronuclei in bone marrow erythrocytes was observed following administration of the test item to mice at up to and including 100 mg/kg bw/day; thus, there was no evidence of any genotoxic activity of the test item under the conditions of this study.