Phosphoric acid, iron(2+) lithium salt (1:1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: genome mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 2012 to 10 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Details on test material:

- Physical state: solid
- Appearance: grey
- Storage condition of test material: room temperature, protected from light and stored in closed bags

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River. Sulzfeld Germany
- Age at study initiation: 6-12 weeks
- Weight at study initiation: male: 25.1-31.7g ; female: 20.3-25.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals were housed (by sex) in IVC cages furnished with Altromin saw fiber bedding.
- Diet: Altromin 1324 maintenance diet for rats and mice (ad libitum)
- Water: ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10 %
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: cotton seed oil;
-Volume: 10 mL/kg bw

Details on exposure:

PREPARATION OF THE TEST MATERIAL
The test material was thoroughly ground in a mortar and diluted in cottonseed oil within 1 hour before treatment. All animals received a single volume ip of 10 mL/kg bw.

Duration of treatment / exposure:

Single ip administration.

Frequency of treatment:

Single test material administration.

Post exposure period:

Sampling of the peripheral blood was carried out on animals 44 hours (all groups) and 68 hours (negative control and highest dose groups) after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (negative control), 400, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 males and 5 females per dose (0, 400, and 1000 mg/kg bw);
7 males and 7 females (2000 mg/kg bw) - 2 animals per sex were reserve animals

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide;
- Justification for choice of positive control(s):
- Route of administration: ip
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Doses were selected on the basis of the results obtained from the preliminary toxicity test.

TREATMENT AND SAMPLING TIMES
At the beginning of the experiment the animals were individually weighed and the administered volume adjusted to the animal's body weight. The animals received the test material once ip. Sampling of the peripheral blood was carried out on animals 44 and 68 hours after treatment.

DETAILS OF SLIDE PREPARATION
Blood was obtained from the animal's tail vein following incision. Blood cells were immediately fixed in ultracold methanol. Before analysis (at least 16 hours after fixation), fixed blood cells were washed in Hank's balanced salt solution, centrifuged at 600 x g for 5 minutes and the supernatant discarded. Blood cell populations were discriminated using specific antibodies against CD71 (expressed only at the surface of immature erythrocytes) and CD61 (expressed at the surface of platelets) and DNA content of micronuclei was determined by the use of a DNA specific stain.

METHOD OF ANALYSIS
Evaluation of all samples, including those of positive and negative controls, was performed using a flow cytometer. Anti-CD71 antibodies were labelled with fluorescein-isothiocyanate, anti-CD61 antibodies were labelled with phycoerythrin. Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensity were recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI, respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test material the ratio between immature and mature erythrocytes was determined.

Evaluation criteria:

The criteria for determining a positive result are as follows:
- dose related increase of micronucleated cells and/or
- biologically relevant increase in the number of micronucleataed cells for at least one of the dose groups

The criteria for a negative result are as follows:
- no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level

Acceptance criteria
The data generated are considered acceptable if:
- at commencement of the study, the weight variation of animals does not exceed ± 20% of the mean weight of each sex
- the background frequency of micronucleated cells is in the normal range as reported in literature or within the laboratory's historical range
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.

Statistics:

The non-parametric Mann-Whitney Test was performed to verify the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: After receiving a single ip dose of test material, three male and three female animals showed signs of toxicity, such as reduction in spontaneous activity, constricted abdomen, piloerection, bradykinesia, opisthotonos and half eyelid closure. Due to the results obtained, 2000 mg/kg bw was chosen as the maximum tolerable dose in the main experiment.

RESULTS OF DEFINITIVE STUDY
- Toxicity:
The animals treated with 2000 mg/kg bw showed slight to moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, eye closure, kyphosis and opisthotonos. The animals treated with 1000 and 400 mg/kg bw showed increasingly less toxicity, respectively.
- Body weights:
The weight variation of the animals did not exceed ± 20% of the mean weight of each sex.
- Relative PCE:
The negative controls (44 h, 68 h) were within the range of the historical control data of the negative control (1.19 - 3.97). The mean values noted for the 44 hour negative control were 3.40 for the male and 1.81 for the female mice. The mean values detected for the 68 hour negative control were 3.26 for the male and 2.02 for the female mice.
Mice treated with 400 mg/kg bw test material showed mean values of the relative PCE of 2.96 (males) and 1.64 (females). These values are decreased from the negative controls, but this decrease is not statistically significant.
Mice treated with 100 mg/kg bw showed mean values of relative PCE of 2.70 (males) and 1.78 (females). These values are decreased from the negative controls, but the values were within the range of historical control data.
Mice treated with 2000 mg/kg bw (44 hour treatment) showed mean values of 2.74 (males) and 1.41 (females). These values are decreased from the negative controls.
Mice treated with 2000 mg/kg bw (68 hour treatment) showed mean values of 1.56 (males) and 0.48 (females). These values are decreased from the negative controls. Additionally, the value observed in the female group was significantly reduced compared to the corresponding control and to the historical control.

- Micronucleated polychromatic erythrocytes:
The negative controls (44 h and 68 h) evaluated were within the range of the historical control data of the negative control (0.08 - 0.43%). The mean values of micronuclei observed for the negative control (44 h) were 0.24% for male and for female mice. The mean values for the 68 h negative control were 0.32% for the male and 0.20% for the female mice.
The mean values of micronuclei observed after treatment with 400 mg/kg bw test material were 0.26% for male and 0.19% for female mice. The value observed in male mice was slightly increased compared to the corresponding negative control. The value observed in female mice was slightly reduced as compared to the corresponding negative control but this increase/decrease was not statistically significant. Additionally, both values were within the historical negative control data.
The mean values for the animals treated with 1000 mg/kg bw dose group were 0.19% for male and 0.25% for female mice. The value observed in the female group was within the range of the corresponding negative control. The value observed in the male group was slightly decreased compared to the corresponding negative control. However, both values were within the range of the historical negative control data.
The dose group treated with 2000 mg/kg bw test material (44 h treatment) showed mean values of 0.35% for the male and 0.38% for the female mice. The values observed in the male and female groups were increased compared to the corresponding negative control, but these increases were not statistically significant. Additionally, both values were within the range of the historical negative control data.
The mean values observed for the 2000 mg/kg bw animals for the 68 hour treatment were 0.32% for the male and 0.29% for the female mice. These values were within the range of the corresponding negative control.
No biologically relevant increase of micronuclei was found after treatment with the test material in any of the dose groups evaluated.
No statistically significant increases (p<0.05) of cells with micronuclei were noted in the dose groups of mice treated with the test material.

Positive control:
Cyclophosphamide (40 mg/kg bw) induced a statistically significant increase of induced micronucleus frequency (mean percentage of cells with micronuclei was 2.19% for male and 1.88% for female mice. These results demonstrate the validity of the assay.

Any other information on results incl. tables

Dose Group	Concentration [mg/kgbw]	Preparation (h)	Male [%]	Female [%]
Negative Control	0	44	0.24	024
Cyclophosphamide	40	44	2.19	1.88
MTD	2000	44	0.35	0.38
MTD/2	1000	44	0.19	0.25
MTD/5	400	44	0.26	0.19
Negative Control	0	68	0.32	0.20
MTD	2000	68	0.32	0.29

MTD = maximum tolerable dose

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of the study, the test material did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

The potential of the test material to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse was investigated in a study which was conducted under GLP conditions and in accordance with the standardised guideline OECD 474.

During the study the test material was prepared and diluted in cottonseed oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single administration of the test material.

A pre-experiment and a dose of 2000 mg/kg bw was subsequently selected as maximum tolerable dose (MTD). The signs of toxicity were noted .

In the main experiment three dose levels were used covering a range from the maximum tolerable dose to no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

Doses	Concentration (mg/kg bw)
MTD	2000
MTD/2	1000
MTD/5	400

The animals treated with doses of 400 and 1000 mg/kg bw showed only slight signs of systemic toxicity. The animals treated with a dose of 2000 mg/kg bw showed slight and moderate signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, half eyelid closure, eye closure, kyphosis and opisthotonos.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the laboratory control data. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the historical negative control data range.

No biologically relevant increase of micronuclei was found after treatment with the test mateial in any of the dose groups evaluated.

The non-parametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p <0.05) of cells with micronuclei were noted in the dose groups.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a significant increase in micronucleus frequency thereby demonstrating the validity of the assay.

The test material is therefore considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.