Phosphonic acid, monoethyl ester, sodium salt (1:1)

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

Remarks:

(guideline adopted 1996)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(adopted in March 1996)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

at least 4000 PCEs should be scored; slightly younger animals were used

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

(adopted in June 1996)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium triethyl triphosphonate

EC Number:

254-320-2

EC Name:

Aluminium triethyl triphosphonate

Cas Number:

39148-24-8

Molecular formula:

C2H7O3P.1/3Al

IUPAC Name:

aluminum tris(ethyl phosphonate)

Specific details on test material used for the study:

- Purity test date: 13 Sep 1996 (reanalysis: 13 Sep 1998)

Test animals

Species:

mouse

Strain:

other: CD-1 (outbred)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, UK
- Age at study initiation: range-finder: 51 - 66 days, main study: 36 - 43 days
- Weight at study initiation: range-finder: 31 - 39 g (males), 24 - 33 g (females); main study: 24 - 33 g (males), 20 - 26 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: animals were not starved for diet and water prior to dosing
- Housing: animals were housed in groups of no more than 3 animals of the same sex
- Diet: Special Diets Services Ltd, RM1. [E]. SQC., ad libitum
- Water: public water supply, ad libitum
- Acclimation period: range-finder: 15 days, main study: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 42 - 65
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: methyl cellulose (0.5% (w/v))
- Concentration of test material in vehicle:
range-finder: 200, 225 and 250 mg/mL
main study: 53.13, 106.3 and 212.5 mg/mL
- Amount of vehicle (if gavage or dermal): 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

Dosing formulations were prepared freshly by suspending Fosetyl Al (with homogenisation) in methyl cellulose. Further dilutions were made in the vehicle to achieve the respective concentrations needed for gavage. Test article preparations were protected from light, mixed by inversion during dosing and used within two hours of formulation.
Analytical analysis verified accuracy of the dosing solutions prepared for the main study (Analytical report CLE 198/112-01F, 1997, Covance). Achieved concentrations ranged from 108 - 130% of the nominal concentrations (low-dose: 108 and 110%, mid-dose: 104 and 112% and high-dose: 127 and 130%) and were thus higher than the nominal concentrations. However, as the signs of toxicity were not unacceptably severe in high-dose animals according to the authors, the study was considered as valid although 4 high-dose females died after administration of the test substance.

RANGE-FINDING STUDY
Initially, a range-finding study was performed to determine the maximum acceptable dose. Groups of 3 animals/sex were exposed once to 4000, 4500 and 5000 mg/kg bw via gavage. Clinical signs of toxicity and body weight development were observed over a time period of 4 days.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single treatment

Post exposure period:

test article and vehicle exposed mice: 24, 48 and 72 h after treatment
CPA exposed mice: 24 h after treatment

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 063 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

2 125 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

4 250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 (plus 5 spare animals/sex exposed to the highest dose)

Control animals:

yes, concurrent vehicle

Positive control(s):

4 mg cyclophosphamide (CPA)/mL in physiological saline
- Justification for choice of positive control(s): due to the known clastogenic properties, CPA was selected as appropriate positive control
- Route of administration: gavage
- Doses / concentrations: 80 mg/kg bw

Examinations

Tissues and cell types examined:

Tissue: bone marrow
Cell type: bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: range finding study performed to find the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): test article and vehicle treated mice were killed 24, 48 and 72 h after gavage. Animals treated with the positive control substance CPA were killed 24 h after substance administration. Mice were killed in the same order as they were dosed.

DETAILS OF SLIDE PREPARATION: bone marrow was isolated from both femurs from each animal except one animal where only a single femur could be sampled. After centrifugation, one drop of cell suspension was used to prepare a smear on a slide, which was allowed to air-dry followed by fixation for 5 min in absolute methanol. Slides were stained with Giemsa (1:6 diluted (v/v)). Afterwards, slides were washed and mounted with coverslips.

METHOD OF ANALYSIS: 2 slides were analysed off-site by a trained analyst and were scored for polychromatic and normochromatic erythrocytes until at least 1000 cells had been analysed. Counting continued until at least 2000 PCE had been observed.

Evaluation criteria:

Evaluation criteria
The test was considered positive if:
1. a statistically significant increase in the frequency of micronucleated PCE occurred at least at one dose, at one sampling time
2. the frequency of micronucleated PCE at such point exceeded the historical control range.
Increased but statistically insignificant increases in frequencies of micronucleated PCE at one samling time in conjunction with either 1 or 2 of the above concentrations at another sampling time were to be cause for additional testing or analysis.

Acceptability
The assay was considered valid if the following criteria were met:
1. the heterogeinity Chi square test provided evidence of acceptable variability between animals within a group, and
2. the incidence of micronucleated PCE in vehicle control groups fell within or close to the historical vehicle control range
3. at least 8 animals (males plus females) out of each group at each kill time were available for analysis
4. the positive control chemical (CPA) induced a statistically significant increase in the frequency of micronucleated PCE.

Statistics:

The ratio of PCE/NCE for each animal and the mean for each group was calculated. Further, the individual and group mean frequencies of micronucleated PCE/1000 were determined. For each group, inter-individual variation in the number of micronucleated PCE was estimated by means of a heterogeneity Chi square test. The numbers of micronucleated PCE in each treated group (males and females, separately and combined) were then compared with the numbers in vehicle control groups by using a 2x2 contingency table to determine Chi square. Probability values of p ≤ 0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

1063 mg/kg bw: 1 unscheduled death, no clinical signs of toxicity; 4250 mg/kg bw: 4/5 females died prior to scheduled sampling and signs of clinical toxicity were observed

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 4000 - 5000 mg/kg bw
- Clinical signs of toxicity in test animals:
5000 mg/kg bw: 1 female was killed in extremis on Day 2 post dosing. Further, several signs of clinical toxicity were obsered including lethargy, prostation, abnormal breathing, eye closure and sunken eyes.
4500 mg/kg bw: 3 males were found dead on Day 1 post dosing. Further, test animals revealed signs of lethargy, tremors, coldness and abnormal breathing.
4000 mg/kg bw: No mortality occured and no clinical signs of toxicity were observed.
-Observation time: Animals were observed for mortality and clinical signs over a period of 4 days.
- Other: Based on the determined mortality and clinical symptoms, 4250 mg/kg bw were established as highest acceptable dose for the main study.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
1063 mg/kg bw: 1 female died prior to the scheduled sampling time. No clinical signs of toxicity were observed.
2125 mg/kg bw: No mortality occured and no clinical signs of toxicity were observed.
4250 mg/kg bw: 4 females died one day after dosing prior to the scheduled sampling time. Further, test animals showed signs of lethargy, hunched posture, eye closure and tremors. To enable inclusion of at least 8 animals per group (males and females) and each sampling time point in the analysis according to the acceptability criteria, the spare females treated with the highest dose were included in the analysis.
- Induction of micronuclei (for Micronucleus assay): Group mean frequencies of micronucleated PCE were comparable among the groups and were not statistically significant different by Chi square test except the mean frequency of the low-dose group determined at the 24 h sampling time point which was statistically decreased compared to the control value. Moreover, a slight but insignificant increase in micronucleated PCE was observed in females at the 48 h sampling time point (0.2, 0.63, 0.4 and 1.0 for control, low-, mid- and high-dose animals). As no dose-dependency was observed, these effects were interpreted as incidental (please refer to Table 2 and 3).
- Ratio of PCE/NCE (for Micronucleus assay): The ratios of PCE/NCE were similar between test and control animals. A slight but not statistically significant increase in the PCE/NCE ratio was observed in females at the 48- and 72 h sampling time points in all dose groups (please refer to Table 2).
- Statistical evaluation: No statistically significant differences were observed in any evaluated parameter among the test groups except the the mean frequency of micronucleated PCE of the low-dose group determined at the 24 h sampling time point which was statistically decreased compared to the control value (please refer to Table 3). As no dose-dependency was observed, the effect was interpreted as incidental (please refer to Table 3).

Any other information on results incl. tables

Table 1: Results of the in vivo micronucleus assay in male mice.

 

			Mean PCE / NCE at sampling time			Total micronuclei per 1000 PCE at sampling time
Exp group	Number of animals	Dose [mg/kg bw]	24 h	48 h	72 h	24 h	48 h	72 h
Vehicle control (methyl cellulose (0.5% (w/v)))	5	0	1.03	0.86	0.90	0.5	0.5	0.6
Positive control (CPA)	5	80	0.6			24.8
Fosetyl Al	4	1063	1.0	0.97	1.13	0.2	0.4	0.7
Fosetyl Al	5	2125	1.41	0.69	0.97	0.2	0.2	0.3
Fosetyl Al	5	4250	0.85	1.0	1.23	0.5	0.5	0.1

 

 

Table 2: Results of the in vivo micronucleus assay in female mice.

 

			Mean PCE / NCE at sampling time			Total micronuclei per 1000 PCE at sampling time
Exp group	Number of animals	Dose [mg/kg bw]	24 h	48 h	72 h	24 h	48 h	72 h
Vehicle control (methyl cellulose (0.5% (w/v))	5	0	0.78	0.83	0.88	0.7	0.2	0.3
Positive control (CPA)	5	80	0.63			14.9
Fosetyl Al	5*	1063	0.77	1.06	1.12	0.10	0.63	0.5
Fosetyl Al	5	2125	0.85	0.97	1.43	0.2	0.4	0.3
Fosetyl Al	5	4250	0.78	1.24	1.5	0.9	1.0	0.4

*: samples from 4 animals were collected for the 48 h sampling time point

 

Table 3: Results of the in vivo micronucleus assay – mean values per treatment group.

 

Treatment group	Dose [mg/kg bw]	Sampling time [h]	Mean frequency of micronucleated PCE per 1000	PCE/NCE ratio
Vehicle control	0	24	0.6	0.91
		48	0.35	0.845
		72	0.45	0.89
Test substance	1063	24	0.15*^t	0.885
		48	0.50	1.015
		72	0.60	1.125
	2125	24	0.2	1.13
		48	0.3	0.83
		72	0.3	1.2
	4250	24	0.7	0.815
		48	0.75	1.12
		72	0.25	1.365
Positive control (CPA)	80	24	19.85**	0.615

*statistically significant (p ≤ 0.05)

**statistically significant (p ≤ 0.001)

^t: represents a statistically decrease in micronucleated cells

Applicant's summary and conclusion

Conclusions:

In this in vivo erythrocytes micronucleus study performed according to OECD 474 and in compliance with GLP, the test substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 4250 mg/kg bw/day. Thus, the test substance was concluded not to be clastogenic.