Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1997-02-14 - 1997-03-13
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): IDS, Na-Salz = Iminodisuccinate, Na-salt or Sodium-iminodisuccinate
- Molecular formula (if other than submission substance): C8H7NO8Na4
- Molecular weight (if other than submission substance): 337.1 [g/mol]
- Structural formula attached as image file (if other than submission substance): see Fig.1
- Substance type: chelate
- Physical state: white powder
- Analytical purity: 67,2 %
- Composition of test material, percentage of components: 67.2% IDS, Na-Salz, 10.1% DL-Asparic acid, Na-salt, 8.3% H20, 7.8% Fumaric acid, Na-salt, 4.2% DL-Malic acid, Na-salt, 1.1% Sodium hydroxide, 0.9% Maleinic acid, Na-salt (analytical result dated February 27, 1997)
- Purity test date: February 27, 1997
- Lot/batch No.: SAV B 0004
- Expiration date of the lot/batch: January 14, 1998
- Storage condition of test material: at room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - mice - strain Hsd/Win: NMRI,
- Source: Harlan Winkelmann GmbH, Borchen
- Age at study initiation: young adult male and virgin female mice, thus approximately 6 to 12 weeks of age
- Weight at study initiation: initially 36-43 g (male) and 27-33 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: singly in type I cages, with a bedding of soft wood granules, type S 8/15 \J. Rettenmaier & Söhne, Füllstoff-Fabriken, Ellwangen-Holzmühle), identified by cage marks
- Diet (e.g. ad libitum): ad libitum, Altromin 1324 Standard Diet
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: at least 5 days

On the day of arrival, (February 3, 1997), the health of the animals was appraised before acclimatizing them to the housing conditions for a period of at least five days. Only healthy animals without symptoms were used in the study.
The feed was offered in troughs. Water was provided in polycarbonate bottles, 300 mL volume

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23°C
- Humidity (%): 40-49% mean relative humidity
- Air changes (per hr): about ten times per hour
- Photoperiod (hrs dark / hrs light): twelve hours of electrical lighting daily (6.00 hours to 18.00 hours, about 500 lux),

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: well known vehicle, no justification needed
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
IDS, Na-Salz was dissolved in deionized water and fox-med a clear, light-yellow solution.
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:
max 2 days
Frequency of treatment:
Each respective substance was administered once
Post exposure period:
14, 24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
1500 mg/kg bw
Basis:

No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): is a proven cytostatic agent and known clastogen with bifunctional alkylation action
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Schmid's method was used to produce the smears.
At least 1 intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and the lower leg. The femur was separated from muscular tissue. The lower-leg stump, incl. the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument, making visible a small opening in the bone-marrow channel. A suitable tube was filled with sufficient foetal calf serum (FCS). A small amount of FCS was drawn from the tube into a syringe with a thin cannula. The cannula was pushed into the open end of the marrow cavity. The femur was completely immersed in FCS and pressed against, the wall of the tube, to prevent its slipping off. The contents were then flushed several times and the bone marrow was passed into the FCS as a fine suspension. Finally, flushing might be repeated from the other end, after it had been opened. The tube containing the FCS and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for 5 min. The supernatant was removed with a suitable pipette, leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well-cleaned slide and spread with a suitable object, to allow proper evaluation of the smear. The labelled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.
The smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.
for more information see "Any other information on materials and methods incl. tables"
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:

METHOD OF ANALYSIS:

OTHER:
Evaluation criteria:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes (PE) were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
It is expedient to establish the ratio of polychromatic to normochromatic erythrocytes (NE) for two reasons:
1. Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alteration of this ratio may show that the test compound actually leaches the target,
Therefore, the number of NE per 1000 PE was noted. If the ratio for a single animal amounts to distinctly more than 3000 NE per 1000 PE, 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 PE/NE 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 NE per 1000 PE, the number of NE showing micronuclei was also established. This information permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated PE, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated NE, without a preceding increase in micronucleated PE, is irrelevant to the assessment of a clastogenic effect, since NE originate from PE. Before an effect can be observed in NE, there must be a much greater increase in micronucleated PE, due to the "dilution effect" of the "old" cells, i.e. NE already present at the start of the test, and this effect would have been observed previously.
Statistics:
The IDS, Na-Salz group (s) with the highest mean (provided this superseded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric 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 (Is ranges) were calculated for all the means.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Remarks:
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 1500 mg/kg IDS, Na-salt, with respect to the incidence of micronucleated polychromatic erythrocytes.
Toxicity:
yes
Remarks:
After a single i.p. administration of 1500 mg/kg IDS, Na-salt, animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, high-stepping gait, sounds + difficulty in breathing. 4 Deaths reported.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1250, 1500, 2500 and 5000 mg/g bw
- Clinical signs of toxicity in test animals: The following symptoms were recorded for up to 48 hours, starting at 1250 mg/kg: apathy, roughened fur, pallor, staggering gait, sternal recumbency, spasm, leaping spasm, high-stepping gait, sounds, difficulty in breathing, slow breathing and slitted eyes. In addition, 2 of 5 animals died in the 1500 mg/kg group and ail animals died in the 2500 and 5000 mg/kg groups.
- Other: based on these results, 1500 mg/kg IDS, Na-Salz was chosen as MTD for this test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 1500 mg/kg IDS, Na-Salz, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.2/1000 (ls=1.4) in the negative control, and 2.9/1000 (ls=1.7), 2.0/1000 (ls=1.4) and 1.9/1000 (ls=1.3) in the IDS, Na-salz group.
- Ratio of PCE/NCE (for Micronucleus assay): the ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with IDS, Na-Salz, being 1000: 714 (ls =144) in the negative control, 1000: 1585 (ls = 506) in the 16 hours group, 1000: 1427 (ls=653) in the 24 hours group and 1000: 1575 (ls=816) in the 48 hours group. Relevant variations were thus noted.
- Appropriateness of dose levels and route: given
- Statistical evaluation: non-parametric Wilcoxon ranking test

Any other information on results incl. tables

Toleration by the Animals

After a single intraperitoneal administration of 1500 mg/kg IDS, Na-Salz, treated animals showed the following compound-related symptoms until sacrifice: apathy, roughened fur, spasm, leaping spasm, high-stepping gait, sounds and difficulty in breathing. Four of 40 treated animals died during the test period, due to the acute toxicity of 1500 mg/kg IDS, Na-Salz. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic Evaluation

Table 2 -6 show the individual results and Table 7 shows the mean results of the test groups for direct comparison. Concerning the assessment of the clastogenic potential of IDS, Na-Salz, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with IDS, Na-Salz, being 1000: 714 (ls =144) in the negative control, 1000: 1585 (ls = 506) in the 16 hours group, 1000: 1427 (ls=653) in the 24 hours group and 1000: 1575 (ls=816) in the 48 hours group. Relevant variations were thus noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 1500 mg/kg IDS, Na-Salz, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.2/1000 (ls=1.4) in the negative control, and 2.9/1000 (ls=1.7), 2.0/1000 (ls=1.4) and 1.9/1000 (ls=1.3) in the IDS, Na-salz group.

Table 2 Results of Micronucleus Test With IDS, Na-Salz
Negative control
Sacrifice 24 hours after treatment
random number and sex number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

13 (male) 1000 828 4.8 3
17 (male) 1000 649 1.5 5
18 (male) 1000 665 0 3
21 (male) 1000 736 2.7 2
30 (male) 1000 558 1.8 3
35 (female) 1000 687 1.5 1
39 (female) 1000 1003 1.0 1
40 (female) 1000 547 3.7 0
44 (female) 1000 609 1.6 2
46 (female) 1000 854 1.2 2
Mean 1000 714 2.0 2.2
ls   144 1.4 1.4

.

Table 3 Results of Micronucleus Test With IDS, Na-Salz
(after acute intraperitoneal treatment with 1500 mg/kg;
Sacrifice 16 hours after treatment
random number and sex number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

10 (male) 1000 1910 1.0 3
11 (male) 1000 1935 0.5 4
16 (male) 1000 1961 2.5 6
19 (male) 1000 2086 1. 4 2
20 (male) 1000 2223 3. 6 4
34 (female) 1000 1019 2.9 0
37 (female) 1000 1648 1.2 2
42 (female) 1000 1013 1.0 3
45 (female) 1000 999 1.0 4
47 (female) 1000 1057 0.9 1
Mean 1000 1585 1. 6 2.9
ls   506 1.0 1,7

.

Table 4 Results of Micronucleus Test With IDS, Na-Salz
(after acute intraperitoneal treatment with 1500 mg/kg)
Sacrifice 24 hours after treatment
random number and sex number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

3 (male) 1000 2539 1.6 2
6 (male) 1000 532 1.9 3
9 (male) 1000 1039 1.9 3
12 (male) 1000 1637 1.8 0
29 (male) 1000 1104 0 4
31 (female) 1000 2416 1.7 3
36 (female) 1000 1520 0.7 1
43 (female) 1000 1560 0 3
55 (female) 1000 1144 0 1
57 (female) 1000 779 1.3 0
Mean 1000 1427 1.1 2 . 0
ls   653 0.8 1.4

.

Table 5 Results of Micronucleus Test With IDS, Na-Salz-
(after acute intraperitoneal treatment with 1500 mg/kg)
Sacrifice 40 hours after treatment
random number and sex

number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

8 (male) 1000 1961 0 2
14 (male) 1000 1093 0.9 4
15 (male) 1000 1022 0 2
22 (male) 1000 720 1.4 0
24 (male) 1000 1435 3.5 3
48 (female) 1000 2462 2.8 2
49 (female) 1000 1758 1.1 0
51 (female) 1000 796 0 2
53 (female) 1000 3312 1.2 3
56 (female) 1000 1195 0.8 1
Mean 1000 1575 1.2 1.9
ls   818 1.2 1.3

.

Table 6 Results of Micronucleus Test With IDS, Na-Salz
Positive Control Cyclophosphamide, 20 mg/kg i.p.
Sacrifice 24 hours after treatment
random number and sex number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

1 (male) 1000 535 0 28
7 (male) 1000 794 3.8 25
23 (male) 1000 586 1.7 26
27 (male) 1000 570 1.8 29
28 (male) 1000 593 3.4 17
32 (female) 1000 583 1.7 11
33 (female) 1000 570 0 17
38 (female) 1000 640 3.1 14
52 (female) 1000 658 3.0 10
60 (female) 1000 611 0 8
Mean 1000 614 1.8 18.5
ls   73 1.5 7.9

.

Table 7 Summary of Results of Micronucleus Test With IDS, Na-Salz
(after acute intraperitoneal treatment with 1500 mg/kg body weight)
experimental groups

number of evaluated poly-chromatic erythrocytes

number of normo-

chromatic erythrocytes per 1000 poly-

chromatic erythrocytes

micronucleated cells per 1000

normo-

chromatic erythrocytes

poly-

chromatic erythrocytes

negative control 10,000 714 2.0 2.2
± 144 ± 1.4 ± 1.4
test substance 16 hours 10,000 1585* 1. 6 2.9
± 506 ± 1.0 ± 1.7
test substance 24 hours 10,000 1427 1.1 2.0
+ 653 ± 0.8 ± 1.4
test substance 48 hours 10,000 1575 1.2 1.9
+ 818 ± 1.2 ± 1.3
positive control CP 20 mg/kg 10,000 614 1. 8 18 . 5*
±   7 3 ± 1.5 ± 7.9
*p<0.01 in non-parametric Wilcoxon ranking test

Similarly, there could be no biologically significant variation between the negative control and IDS, Na-Salz 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 18.5/1000 (ls=7.9), 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: 614 (ls=73) , as against 1000: 714 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.

Assessment

Normally, cells with micronuclei (Howell-Jolly bodies) occur in polychromatic erythrocytes with an incidence of up to approximately 3.0/1000. The increase in micronucleated polychromatic erythrocytes, due, for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model. The results with IDS, Na-Salz gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 1500 mg/kg. The known mutagen and clastogen, cyclophosphamide, had a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg body weight. The number of micronucleated polychromatic erythrocytes increased to a biologically relevant degree. The number of micronucleated norrnochromatic erythrocytes did not increase relevantly in any of the groups. It is of further interest to establish the number of norrnochromatic cells, to learn whether the ratio of polychromatic to norrnochromatic erythrocytes was altered by treatment. The ratio did vary to a biologically relevant degree in the IDS, Na-Salz groups in comparison to the negative control. Cyclophosphamide did not change this ratio.

In conclusion, there was no indication of a clastogenic effect of an intraperitoneal dose of 1500 mg/kg IDS, Na-Salz in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no indication of a clastogenic effect of an intraperitoneal dose of 1500 mg/kg IDS, Na-Salz in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.
Executive summary:

The micronucleus test was employed to investigate IDS, Na-Salz (CAS 144538 -83 -0) in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts (Herbold, 1997, OECD 474). The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. The treated animals received a single intraperitoneal administration of IDS, Na-Salz or cyclophosphamide. The femoral marrow of groups treated with IDS, Na-Salz was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of IDS, Na-Salz and the positive control, cyclophosphamide, were 1500 and 20 mg/kg body weight, respectively.

The animals treated with IDS, Na-Salz showed symptoms of toxicity after administration. Four of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 1500 mg/kg IDS, Na-Salz. There was an altered ratio between polychromatic and normochromatic erythrocytes. However, the frequency of micronucleated immature (polychromatic) erythrocytes is the principal endpoint and the number of mature (normochromatic) erythrocytes in the peripheral blood that contain micronuclei among a given number of mature erythrocytes can only be used as the endpoint of the assay when animals are treated continuously for 4 weeks or more. The increase in micronucleated polychromatic erythrocytes, due, for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model. The results with IDS, Na-Salz gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 1500 mg/kg. Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with miconuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.

In conclusion, there was no indication of a clastogenic effect of an intraperitoneal dose of 1500 mg/kg IDS, Na-Salz in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.