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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-11-29 - 2005-02-01
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Hill formula: C6H9N4O3P CAS formula: C6H9N4O3P
Test material form:
solid: crystalline
Details on test material:
Name:P 101/04
Chemical name: N-(2-Nitrophenyl)phosphoric triamide
Formula: C6H9N4O3P
Description: amber-coloured crystalline solid with slight odour like bitter almond
Purity: > 99 %
Solubility in water: approximately 2 g/l

Test animals

Details on test animals or test system and environmental conditions:
Species / Strain: Mouse, NMRI
Sex:male, female (nulliparous, non-pregnant)
Supplier:Charles River Wiga GmbH, D-97320 Sulzfeld
Hygiene status upon supply: SPF
Age at start of acclimatisation: 6-8 weeks
Acclimatisation:7 days before start of dosing (pre-experiment for toxicity) 14 days before start of dosing (main experiment)
Mean body weight on day of administration:Males:31.4 g± 1.7g(5.4%) n = 25; Females: 27.5 g± 1.4 g (5.3%) n=25

Administration / exposure

Route of administration:
oral: gavage
0.5 % (m/v) solution of Tylose MH 1000 in deionised water
Details on exposure:
oral administration using a metal catheter, single dose, 10 ml/kg body weight
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Doses / concentrations
Doses / Concentrations:
2000 mg/kg bw
actual ingested
No. of animals per sex per dose:
5 males and 5 females
Positive control(s):
Cyclophosphamide (CPA)


Tissues and cell types examined:
Bone Marrow Smears
Details of tissue and slide preparation:
Preparation of Bone Marrow Smears

At the appropriate termination time, the animals were killed by cervical dislocation.

Bone marrow was removed from the femurs using 1 ml of fetal calf serum, each, sedimented using centrifugation at 1200 Rpm for 10 minutes (approximately 700 g) and smears prepared on microscope slides. The smears were aged for approximately 24 hours before staining with May-Grunwald/Giemsa solution. After this staining the polychromatic erythrocytes (PCE) appear bluish, normochromatic erythrocytes (NCE) appear pink to yellowish and micronuclei appears reddish-violet.

Evaluation criteria:
All slides were coded before scoring and scored blind. A minimum of 2000 polychromatic erythrocytes (PCE) was scored for the presence of micronuclei (= MPCE = micronucleated polychromatic erythrocytes) for each animal. The proportion of PCEs among total erythrocytes (PCEs + normochromatic erythrocytes [NCE]) was determined for each animal on the basis of 200 erythrocytes.
The test item is classified mutagenic, if it induces a statistically significant increase at the sampling times of 24 or 48 hours with biological relevance (>0.4 % micronuclei per animal per dose group). A statistically significant increase might require further confirmation by the demonstration of a dose response relationship at the respective sampling time.
Micronucleus scores (MCPE) and the proportion of PCEs among total erythrocytes are presented as individual values. In addition group means and standard deviations are calculated for each sex and experimental group.
The statistical significance compared to the vehicle control were proved by means the Welch t-test (Rasch et al., Verfahrensbibliothek Versuchsplanung und -auswertung, Berlin 1981).
Statistical significance is declared at the 5 % level (one-sided).

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results : negative
Based on the results of the reported study, it is concluded that N-(2-nitrophenyl)phosphoric triamide does not induce an increase of micronuclei in polychromatic erythrocytes of NMRI-mice under the experimental conditions described.

P 101/04 is therefore considered to be non-mutagenic in the mouse bone marrow micronucleus test.
Executive summary:

In a mouse micronucleus test following EC-guideline B.12 under GLP conditions, the ability of N-(2-Nitrophenyl)phosphoric triamide to induce micronuclei was investigated.

Groups of 5 male and 5 female NMRI-mice were exposed to P 101/04 at the limit-test dose of 2000 mg/kg body weight. The test item was administered orally using a metal catheter. A 0.5 % (m/v) solution of Tylose MH 1000 in deionised water (10 ml/kg body weight) served as vehicle control and Cyclophosphamide (CPA) at a dose of 40 mg/kg body weight - also administered orally - was used as positive control. Bone marrow smears were prepared at 24 hours (vehicle control, positive control, dose group) and at 48 hours (vehicle control, dose group) after dosing. Two thousand polychromatic erythrocytes per animal were analysed for the presence of micronuclei. To investigate bone marrow toxicity the proportion of polychromatic erythrocytes among total erythrocytes was evaluated on the basis of 200 erythrocytes. The frequency of micronucleated polychromatic erythrocytes (MPCEs) in the vehicle control group was within the physiological range. Treatment with CPA induced statistically significant increases in the incidence of MPCEs. In none of the experimental groups treated with N-(2-nitrophenyl)phosphoric triamide

an increase in MPCEs or a statistically significant change of the proportion of polychromatic erythrocytes among total erythrocytes was observed. Based on the results of the study reported it is concluded that

N-(2-nitrophenyl)phosphoric triamide

does not induce micronuclei in polychromatic erythrocytes of NMRI-mice under the described experimental conditions.

N-(2-nitrophenyl)phosphoric triamidei

s therefore considered to be non-mutagcnic in the mouse bone marrow micronucleus test.