Fenamiphos

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-08-13 to 1995-10-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

yes

Remarks:

1000 PCE counted per animal; only one dose level tested

Principles of method if other than guideline:

Deviations from OECD 474:
- Only one dose level was used. This level was clearly toxic to the animals. The choice of dose level was based on a pilot test in which animals were exposed to 25 or 100 mg/kg bw of 10 GR. Animals of both dose levels showed toxic symptoms and all animals in the high dose died.
- Only 1000 PCE were scored instead of 2000.
In this case the results for males (5) and females (5) could be taken together (according to the guidelines it is sufficient to test a single sex when there are no substantial differences in toxicity between the sexes).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Fenamiphos

EC Number:

244-848-1

EC Name:

Fenamiphos

Cas Number:

22224-92-6

Molecular formula:

C13H22NO3PS

IUPAC Name:

{ethoxy[3-methyl-4-(methylsulfanyl)phenoxy]phosphoryl}(propan-2-yl)amine

Test material form:

solid: crystalline

Test animals

Species:

mouse

Strain:

other: Hsd/Win: NMR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 - 12 weeks
- Weight at study initiation: Males: 36.0 - 43.0 g (mean: 37.3 g); females 27.0 - 34.0 g (mean: 29.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: The animals were kept singly in type I cages with bedding of soft wood granules
- Diet (ad libitum): Altromin 1324 standard diet (Altromin GmbH, Lage, Germany)
- Water (ad libitum): Tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 - 23.5
- Humidity (%): 40.0 - 70.0
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Deionized water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in deionized water using sonication for 5 minutes, and formed a light-brown turbid suspension. The suspension was stirred with a magnetic mixer during administration and injected intraperitoneally. Cyclophosphamide was dissolved in deionized water and administered in the same way. The negative control received deionized water by the same method. In all of the groups, the administered volume was 10 mL/kg body weight.

Duration of treatment / exposure:

not applicable

Frequency of treatment:

single application

Post exposure period:

16, 24 or 48 h

Doses / concentrations

Dose / conc.:

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

No. of animals per sex per dose:

5 animals per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES: 16, 24 and 48 h
Sacrifice of the negative and positive controls was after 24 hours

DETAILS OF SLIDE PREPARATION:
From each sacrificed animal, one intact femur was prepared. Bone marrow cells were washed out by injecting fetal bovine serum into one end of the dissected epiphyses of each femur. Following centrifugation, excess supernatant was removed and the cells re-suspended in a small volume of serum remaining in the tube. A drop of the suspension was used to make a smear. The smears were dried overnight dried with heat for a short period. The smears were stained automatically. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry before covering.

METHOD OF ANALYSIS:
One slide per animal was examined at 1000-fold magnification by light microscopy. Normally, 1000 polychromatic erythrocytes were counted per animal. The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A relevant, treatment related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority of the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established. This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated polychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones. Before an effect can be observed in normochromatic erythrocytes, there must be a much greater increase in micronucleated polychromatic erythrocytes, due to the "dilution effect" of the "old" cells, i.e. normochromatic erythrocytes already present at the start of the test, and this effect would have been observed previously.

VALIDITY CRITERIA
(1) The frequency of micronucleated polychromatic erythrocytes in the negative control group was within an acceptable range based on the historical control data in the testing facility;
(2) The frequency of micronucleated polychromatic erythrocytes in the positive control group was statistically higher than that of the concurrent vehicle control group.

Evaluation criteria:

A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control.
A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which was within the range of negative controls. In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

Statistics:

The test item groups with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's nonparametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control. The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi2-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The dose selectionof the test item was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 25 mg/kg and 100 mg/kg test substance. The following symptoms were recorded for up to 48 hours, starting at 25 mg/kg: apathy, roughened fur, spasm, extension spasm and leaping spasm. In addition, 5 of 5 animals died in the 100 mg/kg group. Based on these results, 25 mg/kg test subtance was chosen for this test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: 1.7/1000 (1s=1.4, negative contro)l, 1.8/1000 (1s=1.7), 1.1/1000 (ls=1.0) and 1.5/1000 (1s=1.0) in the test item 16, 24 and 48 h groups
- Ratio of PCE/NCE: 1000:1047 (1s=506, negative control), 1000:1027 (1s=268, the 16 hours group), 1000: 865 (1s=208, 24 hours group) and 1000:939 (1s=471, 48 hours group)

After single intraperitoneal administration of 25 mg/kg of the test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

Concerning the assessment of the clastogenic potential of the test item, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test item, being 1000:1047 (1s=506) in the negative control, 1000:1027 (1s=268) in the 16 hours group, 1000: 865 (1s=208) in the 24 hours group and 1000:939 (1s=471) in the 48 hours group. No relevant variations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 25 mg/kg the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.7/1000 (1s=1.4) in the negative control, and 1.8/1000 (1s=1.7), 1.1/1000 (ls=1.0) and 1.5/1000 (1s=1.0) in the the test item groups.
Similarly, there could be no biologically significant variation between the negative control and the test item groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 18.7/1000 (ls=6.7), which represents a biologically relevant increase in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones. No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree [1000:862 (ls=221), as against 1000:1047 in the
negative control]. This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.
All validity criteria were met.

Applicant's summary and conclusion

Conclusions:

There was no indication of a clastogenic effect of an intraperitoneal dose of 25 mg/kg bw the test subtance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Executive summary:

The test item was analysed for its clastogenic potential in the in vivo micronucleus test.

Mice (5/sex/dose) were dosed by intraperitonial injection of the test substance at a dose of 0 or 25 mg/kg bw (equivalent to 2.5 mg test item/kg bw). Deionized water was used as vehicle and cyclophosphamid served as positive control test item. The bone marrow was sampled 16, 24 and 48 h after dosing. 1000 polychromatic erythrocytes (PCE) were counted per animal. The number of normochromatic erythrocytes (NCE) per 1000 polychromatic ones was noted. In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established.

After single intraperitoneal administration of 25 mg/kg of the test substance, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, periodically stretching of body and difficulty in breathing. Their feeding behavior was normal. There were no substance-induced mortalities. No symptoms were recorded for the control groups. No animals died in these groups.

The following PCE/NCE ratios were established: 97%, 115%, 106% for 16, 24 and 48 h after dosing, respectively (the ratio for positive control was 121% and for the negative control 96%). The ratio of polychromatic to normochromatic erythrocytes was not altered by the treatment with the test item. No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 25 mg/kg of the test item, with respect to the incidence of micronucleated polychromatic erythrocytes. Similarly, there could be no biologically significant variation between the negative control and test item groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. All validity criteria were met in this micronucleus test.

It was concluded that there was no indication of a clastogenic effect of an intraperitoneal dose of 25 mg/kg bw of the test item in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.