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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:
Between 20/03/1995 and 21/04/1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD 474 and GLP study. The clinical signs observed in main study were not detailed.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
clinical signs observed are not detailed
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
1994-03-16
Type of assay:
micronucleus assay

Test material

Constituent 1
Test material form:
other: liquid stored at room temperature
Details on test material:
- Substance type: liquid (pale straw-coloured)
- Analytical purity: no data
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Manston, Kent
- Age at study initiation: 5-7 weeks
- Weight at study initiation: males: 21-28 g, females 20-24 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: groups of up to 5 by sex.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21-22 C°
- humidity 47-50%
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Sterile distilled water
- Concentration of test material in vehicle: 21.25, 42.5 and 85 mg/mL for dose levels 212.5, 425 and 850 mg test material/kg respectively
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: 401034
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: freshly prepared as required as a solution in distilled water
Duration of treatment / exposure:
Not applicable (following single gavage, 24 or 48-hour observation period before sacrifice)
Frequency of treatment:
once
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
212.5, 425 and 850 mg test material/kg bw , equivalent to 140.2, 280.5 and 561 mg Active Ingredient (AI)/kg bw respectively.
Basis:
nominal conc.
Animals were dosed at 212.5, 425 and 850 mg test material/ kg following range-finding toxicity study which showed 0/4 deaths at 750 and 850 mg test material/ kg bw, 1/4 deaths at 900 mg test material/kg bw and 2/4 at 1000 mg test material/kg bw.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: cyclophosphamide monohydrate
- Justification for choice of positive control(s): no data
- Route of administration: gavage
- Volume administered: 10 mL/kg b.w

Examinations

Tissues and cell types examined:
Both femurs were dissected out from each animal to remove the proximal epiphysis from each bone. The bone marrow of both femurs from each animal was pooled in order to obtain cell suspension (see Table 7.6.2/2).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: preliminary toxicity test was performed to determine a suitable dose level for use in the micronucleus test; the maximum tolerated dose, i.e. the highest dosage which would be expected to elicit signs of toxicity without producing extreme clinical signs or having a significant effect on survival.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): no data

DETAILS OF SLIDE PREPARATION: dissected femurs were aspirated with foetal calf serum. Bone marrow smears were prepared following centrifugation and re-suspension. Smears were air dried, fixed in absolute methanol and stained in May-Grünwald/Giemsa.

METHOD OF ANALYSIS: The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes/animal.

OTHER: no data
Evaluation criteria:
- A statistically significant dose-related increase in the frequency of micronucleated polychromatic erythrocytes for 24 or 48h kill times relative to control is indicative of a positive mutagenic response.
- A positive response for bone marrow toxicity is indicated when the polychromatic to normochromatic ratio is significantly different from the vehicle control group.
Statistics:
All data were analysed using Student’s t-test (two tailed), and any significant results were confirmed using the one way analysis of variance. The analyses used are considered to be appropriate.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Recorded on in preliminary study: at 900 and 1000 mg test material /kg and above : Premature deaths were seen, and the following clinical signs were observed: hunched posture, lethargy, decreased respiration rate, laboured respiration, ptosis and ataxia.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 750-1000 mg test material/kg bw
- Clinical signs of toxicity in test animals: loss of righting reflex, decreased respiratory rate, laboured respiration, ataxia, lethargy, ptosis, hunched posture, splayed gait, red-stained urine and pallor of the extremities observed at and above 900 mg test material/kg bw.
- Other: premature mortalities at 900 mg test material/kg bw and above
Conclusion: 212.5-850 mg Test material/kg bw selected for defenitive study.
RESULTS OF DEFINITIVE STUDY
- Mortality: 1 female died at 212.5 mg/kg-1 (Sample time: 24 hours) and 2 females died at 850 mg/kg-1 (sample time: 48 hours).
- Induction of micronuclei (for Micronucleus assay): there was no evidence of induction of micronuclei by the test substance See table 7.6.2/3
- Ratio of PCE/NCE (for Micronucleus assay): there was no statistically significant change in the PCE/NCE ratio in any of the test material groups when compared to their concurrent vehicle control groups, however a dose-related reduction in the PCE/NCE ratio in the 24-hour dose groups was observed , this together with the presence of clinical signs and premature deaths would indicate systemic absorption had occurred.
- Appropriateness of dose levels and route: appropriate
- Statistical evaluation: see description above

Any other information on results incl. tables

 Table 7.6.2/3: Results of in vivo micronucleus test with THPC-urea

 

Control

Low dose

Mid dose

High dose

Number of cells evaluated

1378

 1282

 1413

 1424

Sampling time (h)

 

 

24 h

 

Number of erythro-cytes

normo­chromatic

378+/-70

 282+/-66

413+/-68

424+/-63

poly­chromatic

1000+/-0

 1000 +/- 0

 1000 +/-0

 1000+/-0

polychromatic with micronuclei

 0.9+/-1.1 

 0.7 +/-0.7

 1.1+/-1.21

  1.2+/-0.9

Ratio of erythro­cytes

polychromatic / normochromatic

1.73+/-0.53 

 2.71+/-0.86

 1.50 +/-0.51

 1.41+/-0.38

polychromatic with micro­nuclei / normochromatic

 0.02 +/-0.02

 0.03 +/-0.01

 0.03 +/-0.02

 0.03 +/-0.01

 

Control

Low dose

Mid dose

High dose

Number of cells evaluated

1372

 -

 -

 1465

Sampling time (h)

 

 

48h

 

Number of erythro-cytes

normo­chromatic

372+/-0.1

 -

-

465+/-128

poly­chromatic

1000+/-1.1

 -

 -

 1000+/-0

polychromatic with micronuclei

1.1+/-1.2

 -

-

  0.9+/-1.1

Ratio of erythro­cytes

polychromatic / normochromatic

1.78+/-0.57

-

 -

 1.31+/-0.66

polychromatic with micro­nuclei / normochromatic

 0.03 +/-12

 

 

 0.02 +/-0.09

 

- Number of PCE with Micronuclei per 1000 PCE (positive control 24 -hour sampling time): 31.4*** +/- 12.2

- PCE/NCE Ratio (positive control 24 -hour sampling time): 1.79 +/- 0.78

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the test conditions, Tetrakis (hydroxy methyl) phosphonium chloride, oligomeric reaction products with urea (THPC-urea) was considered to be non-genotoxic
Executive summary:

Bone marrow micronucleus assay was conducted in CD-1 mouse according to OECD guidelines 474 (SPL, 1996) and in compliance with GLP. Animals (5/sex/dose) were treated by gavage with test substance identified as Tetrakis (hydroxy methyl) phosphonium chloride, oligomeric reaction products with urea (THPC-urea) at doses of 140, 280 and 561 mg AI/kg b.w. Bone marrow cells were harvested at 24 hours post-treatment for all doses tested groups and at 48 hours for the highest dose group. The water was used as concurrent vehicle negative control. A group of 5 males and 5 females were treated by gavage with cyclophosphamide as concurrent positive control.

There was no statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups, however, a dose-related reduction was observed in the 24 -hour groups, this together with the presence of clinical signs and premature deaths would indicate systemic absorption had occured. Test substance was tested at adequate doses based on a preliminary assay at 495, 591, 594 and 660 mg AI/kg. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.