Neodymium di(2-ethylhexyl) phosphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 14 December 2007 to 29 July 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: the study was performed according to internationally recognised guidelines and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

temperature sometimes outside of the target ranges and analysis of the test item concentration in plasma not performed (deviations not considered to have compromised the validity or integrity of the study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see higher

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see higher

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: at the beginning of treatment the mean bogy weight was 33 g for males (ranging from 29 to 36 g) and 25 g for females (ranging from 23 to 28 g)
- Assigned to test groups randomly: yes, under following basis: by sex
- Fasting period before study: no data
- Housing: by groups in polycarbonate cages
- Diet: free access to Ssniff R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany)
- Water: drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 30 to 70%
- Air changes: at least 12 cycles/hour of filtered non-recycled fresh air
- Photoperiod: 12hrs dark / 12hrs light (07:00 - 19:00)

IN-LIFE DATES: from 19 December 2007 (first day of treatment in the preliminary toxicity test) to 24 January 2008 (Sacrifice of the animals and preparation of bone marrow smears)

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: 026 K 0051 and 126 K 0117 (Sigma, Saint-Quentin-Fallavier, France)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For the main test, the test item was ground to a fine powder using a mortar and pestle, suspended in the vehicle in order to achieve the concentrations of 50, 100 and 200 mg/mL and then homogenized using a magnetic stirrer.
The preparations were made immediately before use.

Duration of treatment / exposure:

2-day period

Frequency of treatment:

two treatments separated by 24 hours

Post exposure period:

24 hours after the last treatment

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000 and 2000 mg/kg/day
Basis:
nominal conc.

No. of animals per sex per dose:

Preliminary toxicity test: 3 male and 3 female mice
Main cytogenetic test: 5 male and 5 female mice (+ 3 male and 3 female mice in the high-dose group as satellite animals for determination of plasma level of the test item)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): recommended in the guideline
- Route of administration: oral
- Doses / concentrations: one treatment at 50 mg/kg/day

Examinations

Tissues and cell types examined:

bone marrow smears (erythrocytes)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
In order to determine the highest dose-level, a preliminary test was performed on a group of six animals (three males and three females). Clinical signs and any mortality were recorded for a period of 48 hours. At the end of this period, the animals were killed by CO2 inhalation in excess.

TREATMENT AND SAMPLING TIMES:
At the time of sacrifice (24 hours after the second treatment), all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum.

DETAILS OF SLIDE PREPARATION:
After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS:
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE (micronucleated polychromatic erythrocytes) must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance (≥ 3 groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Whitney test (two groups) or a Kruskall Wallis test (≥ 3 groups) was performed followed by a Dunn test (if necessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.417, SAS Institute Inc), with a level of significance of 0.05 for all tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg/day
- Solubility: no data
- Clinical signs of toxicity in test animals: no mortality occurred at 2000 mg/kg/day. Two out of three males and two out of three females showed half-closed-eyes 4 hours after the second treatment only.
- Evidence of cytotoxicity in tissue analyzed: not examined
- Rationale for exposure: the oral route was chosen, since it is a possible route of exposure in human
- Harvest times: 24 hours after the second treatment

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: the mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle group.
- Ratio of PCE/NCE: see table below
- Appropriateness of dose levels and route: the top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since non severe toxic effects were observed at 2000 mg/kg/day, this dose-level was selected as the top dose-level for the study. The oral route was chosen, since it is a possible route of exposure in human.
- Statistical evaluation: no statistically significant increase in the frequency of MPE was observed

The PE/NE ratio was significantly higher in the female positive control group when compared to that of the corresponding vehicle group. This effect was not considered of any biological significance.
The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with our historical data.
Cyclophosphamide induced a statistically significant increase in the frequency of MPE (p < 0.05 and p < 0.01 for the males and females respectively) when compared to that of the vehicle group, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Any other information on results incl. tables

Table 1: Results of the cytogenetic test

Sex	Group	Doses (mg/kg/day)	MPE/1000PE (mean±SD)	PE/NE ratio (mean±SD)	Time of sacrifice after the last administration
Males	Vehicle Test item     Cyclophosphamide	- 500 1000 2000 50	1.1± 0.4 0.9 ±0.7 1.4 ± 1.8 0.3 ± 0.3 51.2 ± 11.8 *	0.5± 0.1 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.2 0.6 ± 0.2	24 h
Females	Vehicle Test item     Cyclophosphamide	- 500 1000 2000 50	1.0± 0.6 1.0 ±0.7 1.1 ± 1.0 1.2 ± 0.8 19.2 ± 6.2 **	0.6± 0.2 0.6 ± 0.1 0.7 ± 0.1 0.6 ± 0.1 1.0 ± 0.2 *	24 h

Five animals per group

MPE: Micronucleated Polychromatic Erythrocytes

PE: Polychromatic Erythrocytes

NE: Normochromatic Erythrocytes

SD: standard deviation

Statistical significance: * p < 0.05** ; p < 0.01

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Neodymium tris (di-2-ethylhexyl phosphate) did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells in-vivo by oral route.

Executive summary:

In a bone marrow micronucleus study performed according to OECD 474, EU B.12, EPA OPPTS 870.5395 and GLP (CIT report No.33215 MAS, 2008), scored as validity 1 according to Klimisch criteria, three groups of five male and five female mice were given neodymium tris (di-2-ethylhexylphosphate) in corn oil at dose-levels of 500, 1000 and 2000 mg/kg/day by oral route, over a 2-day period (two treatments separated by 24 hours). Concurrent vehicle control (corn oil) and positive control (cyclophosphamide) were tested in parallel.

The animals were killed 24 hours after the last treatment.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

 

Neither mortality, nor clinical signs were observed in the animals of either sex at any dose-levels.

The mean values of MPE as well as the PE/NE ratio in the groups treated with the test item, were equivalent to those of the vehicle group. The vehicle and positive controls gave appropriate response, consistent with the historical data.

 

Under these experimental conditions, neodymium tris (di-2-ethylhexylphosphate) did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 and 2000 mg/kg/day.

This study is classified as acceptable. It satisfies international guideline requirements for in-vivo micronucleus assay.