N-methyl-P,P-bis(2-methylaziridin-1-yl)phosphinamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no study available

Genetic toxicity in vivo

Description of key information

In vivo bone marrow micronucleus test in mice: positive (OECD 474, GLP, Rel. 1, K)

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 September to 27 December 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 474 without deviation.

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)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: SNPE, France / 106/04
- Description: Whitish solid (viscous liquid at 20 °C)
- Date of receipt: 31 August 2005
- Expiration date of the lot/batch: 28 June 2006 (retest date)
- Purity test date: 26 August 2005

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at 4 °C and protected from humidity

Species:

mouse

Strain:

other: Swiss Ico: OF1 (IOPS Caw)

Details on species / strain selection:

Rodent species generally accepted by regulatory authorities for this type of study.

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: ca. 6 weeks
- Weight at study initiation: Mean body weight was 31.1 g for males (29.3-33.8 g) and 25.2 g for females (23.2-27.9 g).
- Assigned to test groups randomly: Yes; upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
- Housing: Animals were housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust (SICSA, Alfortville, France).
- Diet: A04 C pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30-70 %
- Air changes: At least 12 cycles/hour of filtered non-recycled fresh air.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 27 September to 27 December 2005

Route of administration:

oral: unspecified

Vehicle:

- Vehicle(s)/solvent(s) used: Water for injections
- Concentration of test material in vehicle: 0.5, 1 and 2 mg/mL
- Dose volume: 10 mL/kg bw/day

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- For the main test, the test item was dissolved in the vehicle in order to achieve the concentrations of 2 mg/mL and then homogenized using a magnetic stirrer. Further dilutions were performed to obtain final concentrations of 1 and 0.5 mg/mL. The preparations were made immediately before use.

Duration of treatment / exposure:

Two days

Frequency of treatment:

Two treatments separated by 24 h

Post exposure period:

24 h after the last treatment

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Dose / conc.:

20 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 and 10 mg/kg/day - 5 animals/sex/dose (principal)
20 mg/kg/day - 8 animals/sex/dose # (principal); 3 animals/sex/dose ## (satellite)

# Since no mortality occurred, only five animals of each sex were subjected to bone marrow analysis.
## Satellite animals allocated for determination of plasma level of the test item.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: Cyclophosphamide
- Route of administration: Oral
- Concentration in vehicle: 5 mg/mL
- Dose volume: 10 mL/kg bw
- Doses: 50 mg/kg bw; one treatment
- No. of animals: 5/sex/dose

Tissues and cell types examined:

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).

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Preliminary toxicity test: In order to determine the highest dose-level, a first preliminary test was performed on groups of males and females. Clinical signs and any mortality were recorded for a period of 48 h. At the end of this period, the animals were killed by CO2 inhalation in excess. In view of the high bone marrow toxicity observed in the first main test (rejected experiment) and in order to determine the high dose-level for the repeat main test, a second preliminary test was performed on groups of six animals (three males and three females). Clinical signs and any mortality were recorded over a period of 48 h. At the end of this period, the animals were killed by CO2 inhalation in excess and bone marrow smears were performed. For each animal, the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 500 erythrocytes (PE + NE).

TREATMENT AND SAMPLING TIMES:
Animals were given oral administrations of test item at dose-levels of 5, 10 or 20 mg/kg bw/day, over a 2-day period. The animals of the treated and vehicle control groups were killed 24 h after the last treatment and the animals of the positive control group were killed 24 h after the single treatment. Bone marrow smears were then prepared.

DETAILS OF SLIDE PREPARATION:
At the time of sacrifice, 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. 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.

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 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 (≥ three 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 (≥ three 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.

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF PRELIMINARY TOXICITY TEST
- In order to select the top dose-level for the cytogenetic study, dose-levels ranging from 10 to 1000 mg/kg bw/day were tested. In first instance, selection of the highest dose-level for the main test was performed on the basis of observation of mortality and/or clinical signs (maximum tolerated dose), as follows:
- In males, since the dose-level of 1000 mg/kg bw/day induced mortality (3/3 treated animals) and lower dose-levels of 500 and 750 mg/kg bw/day induced no mortality and showed clinical signs (piloerection, half-closed eyes and sometimes hypoactivity), the latter was retained as the maximum tolerated dose-level. In females, since the dose-level of 500 mg/kg bw/day induced mortality (1/3 treated animals) and lower dose-level of 375 mg/kg bw/day induced no mortality and showed clinical signs (piloerection and half-closed eyes), the latter was retained as the maximum tolerated dose-level. Therefore for the main test, the dose-levels of 187.5, 375 and 750 mg/kg bw/day for males and 93.75, 187.5 and 375 mg/kg bw/day for females were selected.
- Following spreading of bone marrow slides and their quality control, it was found that very high bone marrow toxicity was induced in all test item treated groups of males and females leading to a huge decrease in the number of polychromatic erythrocytes. Therefore micronucleus analysis could not be performed on these treated animals. The treatment was repeated at lower dose-levels and for selection of the highest dose-level bone marrow induced toxicity (PEINE ratio) was also taken into account.
- At 50 mg/kg bw/day, except for piloerection noted in males, no clinical signs and no mortality was induced. Bone marrow analysis showed a very low PE/NE ratio in both males and females (mean value for males and females of 0.07 ± 0.11 versus a historical mean value of 0.5 and 0.7 for males and females respectively).
- At 20 and 10 mg/kg bw/day, no clinical signs and no mortality were noted. Bone marrow analysis showed a mean PE/NE ratio of 0.34 and 0.40, respectively.
- The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects (based on the evaluation of bone marrow toxicity) were observed, the choice of the top dose-level was based on the level of toxicity. Consequently, 20 mg/kg bw/day was selected as the top dose-level for the main test. The two other selected dose-levels were 10 and 5 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY
- No clinical signs and no mortality were observed in the animals of both sexes given 5, 10 or 20 mg/kg bw/day.
- Induction of micronuclei (for Micronucleus assay): For both males and females, the mean values of MPE in the groups treated with the test item, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 6.3-20.2 MPE/1000 PE for males versus 0.1-2.8 MPE/1000 PE for historical mean vehicle control value and 4.2-13.2 MPE/1000 PE for females versus 0.2-1.6 MPE/1000 PE for historical mean vehicle control value. Due to the high variability noted in MPE frequencies in the test item treated groups, the differences observed between these groups and the concurrent vehicle controls were not always statistically significant. However the increase in frequencies of MPE obtained were considered of high biological relevance.
- Ratio of PE/NE (for Micronucleus assay): See Table 7.6.2/1

VEHICLE AND POSITIVE CONTROL RESULTS
- The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.
- Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Table 7.6.2/1: Results of the cytogenetic test: data summary

 

Group	Doses (mg/kg bw/day)	MPE/1000PE		PE/NE ratio		Time of sacrifice after the last administration
		Mean	SD	Mean	SD
Males
Vehicle	-	1.3	0.8	0.5	0.1	24 h
Test item	5	6.3	4.7	0.5	0.2
	10	20.2	26.9	0.5	0.2
	20	15.5	10.4	1.0	0.5
Cyclophosphamide	50	27.8**	8.8	0.8*	0.4
Females
Vehicle	-	1.0	0.4	0.7	0.1	24 h
Test item	5	10.0*	8.3	0.5	0.3
	10	4.2	3.8	0.8	0.1
	20	13.2**	9.2	0.7	0.3
Cyclophosphamide	50	20.1*	3.6	0.9	0.2

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

Conclusions:

Under the experimental conditions, the test item induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24 h interval, at the dose-levels of 5, 10 and 20 mg/kg bw/day.

Executive summary:

In a bone marrow micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, groups of Swiss Ico: OF1 (IOPS Caw) mice (5/sex/dose) were given oral administrations of test item at dose-levels of 0, 5, 10 or 20 mg/kg bw/day, over a 2-day period. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg bw. The animals of the treated and vehicle control groups were killed 24 h after the last treatment and the animals of the positive control group were killed 24 h after the single treatment. Bone marrow smears were then prepared. 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). Preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study.

 

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects (on bone marrow) were observed, the choice of the top dose-level was based on the level of toxicity. Consequently, 20 mg/kg bw/day was selected as the top dose-level for the main test. The two other selected dose-levels were 10 and 5 mg/kg bw/day.

 

For both males and females, the mean values of MPE in the groups treated with the test item, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 6.3-20.2 MPE/1000 PE for males versus 0.1-2.8 MPE/1000 PE for historical mean vehicle control value and 4.2-13.2 MPE/1000 PE for females versus 0.2-1.6 MPE/1000 PE for historical mean vehicle control value. Due to the high variability noted in MPE frequencies in the test item treated groups, the differences observed between the treated groups and the concurrent vehicle controls were not always statistically significant. However the increase in frequencies of MPE obtained were considered of high biological relevance.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

 

Under the experimental conditions, the test item induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24 h interval, at the dose-levels of 5, 10 and 20 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

In a bone marrow micronucleus test conducted according to OECD 474 guideline and in compliance with GLP, groups of Swiss Ico: OF1 (IOPS Caw) mice (5/sex/dose) were given oral administrations of test item at dose-levels of 0, 5, 10 or 20 mg/kg bw/day, over a 2-day period. One group of five males and five females received the positive control test item (Cyclophosphamide) once by oral route at the dose-level of 50 mg/kg bw. The animals of the treated and vehicle control groups were killed 24 h after the last treatment and the animals of the positive control group were killed 24 h after the single treatment. Bone marrow smears were then prepared. 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). Preliminary toxicity tests were performed to define the dose-levels to be used for the cytogenetic study.

 

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since toxic effects (on bone marrow) were observed, the choice of the top dose-level was based on the level of toxicity. Consequently, 20 mg/kg bw/day was selected as the top dose-level for the main test. The two other selected dose-levels were 10 and 5 mg/kg bw/day.

 

For both males and females, the mean values of MPE in the groups treated with the test item, were higher than the concurrent vehicle control groups and out of the vehicle control historical range: 6.3-20.2 MPE/1000 PE for males versus 0.1-2.8 MPE/1000 PE for historical mean vehicle control value and 4.2-13.2 MPE/1000 PE for females versus 0.2-1.6 MPE/1000 PE for historical mean vehicle control value. Due to the high variability noted in MPE frequencies in the test item treated groups, the differences observed between the treated groups and the concurrent vehicle controls were not always statistically significant. However the increase in frequencies of MPE obtained were considered of high biological relevance.

The mean values of MPE as well as the PE/NE ratio for the vehicle and positive controls were consistent with the historical data.Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

 Under the experimental conditions, the test item induced damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24 h interval, at the dose-levels of 5, 10 and 20 mg/kg bw/day.

Justification for classification or non-classification

Positive results were found with the registered substance in an in vivo bone marrow micronucleus test in mice therefore the substance should be classified as category 2 (H341) for germ cell mutagenicity according to Regulation EC 1272/2008 and GHS.