Registration Dossier

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline study with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name: Sa 190
- Formula: C27H53O2N
- Batch No.: #030026-DP-PA10-E01
- CAS No.: 1001161-63-2
- Physical State: liquid with crystalline solid
- Colour: transparent, yellowish
- Purity: > 98%
- Expiry Date: 30 December 2015

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS

- Species: mouse (mus muscufus)
- Strain: NMRI, young healthy adult
- Source: Charles River, 97633 Sulzfeld, Germany
- Number of animals: 5 of each sex per dose group
- Initial age at start of acclimatisation: 6 - 12 weeks
- Age at start of treatment: minimum 7 weeks

The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and undergo an adequate acclimatisation period after arrival. The animals were randomly distributed into test groups and individually marked for identification by tail and ear drawing.

HUSBANDRY

The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
- Housing: 5 animals of identical sex per cage
- Cage type: IVC cage (Polysulphone), Type II L
- Bedding: Altromin saw fiber bedding (Batch: 02102150227)
- Feed: Free access to Altromin 1324 (Batch: 1239) maintenance diet for rats and mice
- Air change: at least 10 x per hour
- Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Environment: temperature 22 ± 3 °C; relative humidity 55 ± 10%; artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Cottonseed oil
Details on exposure:
PREPARATION OF THE TEST ITEM
The test item was grinded thoroughly in a mortar and suspended in Cottonseed oil (Sigma, Batch MKBQ5465V, MKBS9702V) within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw. The solvent was chosen according to its relative non-toxicity for the animals.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
44 and 68 h
Doses / concentrations
Remarks:
Doses / Concentrations:
400, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: CPA (cyclophosphamide)
- CAS No.: 50-18-0
- Supplier: Sigma
- Catalogue No.: C0768
- Batch No.: SLBG4216G
- Dissolved in: physiological saline
- Dosing: 40 mg/kg bw
- Route and frequency of administration: ip, single
- Volume administered: 10 mL/kg bw

The solution was aliquoted and stored at <=-15°C. On day of administration the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled at only a single time. The sampling time for the positive control is 44 h after treatment.

Examinations

Tissues and cell types examined:
Blood cells - erythrocytes
Evaluation criteria:
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECO guideline, the biological relevance as well as the statistical significance of the results are the criteria for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.
Statistics:
For the statistics the nonparametric Mann-Whitney test wass used. However, both biological relevance and statistical significance (at a level of p< 0.05) were considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
other: vehicle served as negative control
Positive controls valid:
yes

Any other information on results incl. tables

PRE-EXPERIMENT

In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. Three male and three female mice received a single dose of 2000 mg/kg bw ip and showed mild toxicity such as reduction of spontaneous activity, constricted abdomen, piloerection, bradykinesia and half eyelid closure. Thus, it could be concluded that the test substance was systemically available. Due to the results obtained in the pre-experiment 2000 mg/kg bw was chosen as maximum tolerable dose (1 MTD) in the main experiment.

Signs of toxicity (2000 mg/kg bw)

 Signs Time post-application / sex (3 male and 3 female mice)
   30 min  1 h  2 h  3 h  4 h  24 h  48 h  72 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity  0  0  0  0  0  0  0  0  3  1  2  1  0  0  0  0
 Constricted abdomen  0  0  0  0  0  0  0  0  1  1  0  0  0  0  0  0
 Piloerection  0  0  0  0  0  0  0  0  2  2  0  1  0  0  0  0
 Half eyelid closure  1  0  1  0  0  0  0  0  3  3  2  0  0  0  0  0
 Bradykinesia  0  0  0  0  0  0  0  0  1  1  0  0  0  0  0  0

MAIN EXPERIMENT

Toxicity

2000 mg/kg bw was tested as the maximum tolerable dose (1 MTD) in the main experiment. The volume administered ip was 10 mL/kg bw.

All animals treated with the highest dose (1 MTD) showed mild toxic effects. After 24 h no toxic symptoms were observed anymore in male as well as female animals. The signs of toxicity noted were reduction of spontaneous activity, bradykinesia and half eyelid closure. Animals treated

with 0.5 MTD and 0.2 MTD showed no signs of toxicity.

Signs of toxicity (2000 mg/kg bw)

 Signs Time post-application / sex (5 male and 5 female mice)
   30 min  1 h  2 h  3 h  4 h  24 h  44 h  68 h
   m  f  m  f  m  f  m  f  m  f  m  f  m  f  m  f
 Red. spont. activity  5  5  5  5  5  5  5  0  5  0  0  0  0  0  0  0
 Half eyelid closure  5  5  5  5  0  0  0  0  0  0  0  0  0  0  0  0
 Bradykinesia  5  0  5  5  0  0  0  0  0  0  0  0  0  0  0  0

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (44 h, 68 h) were within the range of the historical negative control data control (0.88% - 4.30%). The mean values noted for the 44 h negative control were 1.47% (male mice) and 1.43% (female mice). The mean values detected for the 68 h negative control were 2.17% (male mice) and 1.47% (female mice).

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.93% (male and female mice). The mean values observed in both groups were increased as compared to the corresponding negative control. However, these increases were not statistically significant.

The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 1.97% (male mice) and 1.07% (female mice). The value observed in the male group was increased but this increase was not statistically significant. The value observed in the female group was decreased as compared to the corresponding negative control but this decrease was not statistically significant.

The animals who received 1 MTD (44 h treatment) showed mean values of 1.69% (male mice) and 1.63% (female mice). The values observed in both groups were increased as compared to the corresponding negative control. However, these increases were not statistically significant.

The animal group which was treated with 1 MTD (68 h treatment) showed mean values of the relative PCE of 2.90% (male mice) and 2.61% (female mice). The values observed in both groups were increased as compared to the corresponding negative control. However, these

increases were not statistically significant.

The decrease and/or increase of PCE values in treated animals compared to control animals is a hint of a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, at least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (44 h and 68 h) evaluated were within the range of the historical negative control data (0.10 – 0.33%). The mean values of micronuclei observed for the negative control (44 h) were 0.19% (male mice) and 0.15% (female mice). The mean values of the 68 h negative control were 0.23% (male mice) and 0.15% (female mice).

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.22% (male mice) and 0.18% (female mice). The values observed in both groups were within the range of the corresponding negative as well as historical negative control data.

The mean values noted for the 0.5 MTD dose group were 0.22% (male mice) and 0.18% (female mice). The values observed in both groups were within the range of the corresponding negative as well as historical negative control data.

The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.28% (male mice) and 0.18% (female mice). The value observed in the male group was increased compared to the corresponding negative control, but this increase was not statistically significant. Additionally, the value was within the range of the historical negative control data. The value observed in the female group was within the range of the corresponding negative as well as historical negative control data.

The mean values observed for the 1 MTD (68 h treatment) were 0.25% (male mice) and 0.20% (female mice). The value observed in the male group was within the range of the corresponding negative as well as historical negative control data. The value observed in the female group was increased as compared to the corresponding negative control but this increase was not statistically significant. Moreover, the value was within the range of the historical laboratory control data.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a statistically significant increase in the micronucleus frequency (mean percentage of cells with micronuclei was 1.95% for male and 1.36% for female mice. This demonstrates the validity of the assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Sa 190 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, Sa 190 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.
Executive summary:

This study was performed to investigate the potential of Sa 190 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, which is the endpoint of this test to assess genotoxicity.

The test item was grinded thoroughly in a mortar and suspended in Cottonseed oil. The volume administered ip was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after a single application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 420, OECD 423). Based on the outcome of the dose range finding study, a dose of 2000 mg/kg bw was selected as maximum tolerable dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerable dose to little or no toxicity. The following dose groups were selected based on the toxicity observed in the pre-experiment:

 Doses  Concentration [mg/kg bw]
 1 MTD  2000
 0.5 MTD  1000
 0.2 MTD  400

Animals treated with doses of 0.2 MTD and 0.5 MTD showed no signs of systemic toxicity. Animals treated with a dose of 1 MTD showed mild signs of systemic toxicity such as reduction of spontaneous activity, bradykinesia and half eyelid closure.

The observation of systemic toxicity in the pre- as well as in the main-experiment demonstrates that Sa 190 was systemically available after intraperitoneal application and thus was able to reach the target organ.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls (44 h, 68 h) were within the range of the historical negative control data. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the corresponding and historical negative control data range, except the value observed for the 1 MTD male group (44 h) and 1 MTD female group (68 h). These values were increased as compared to the corresponding negative control. However, these increases were not statistically significant and both values were within the range of the historical negative control data. Therefore they were regarded as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the assay.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Sa 190 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.

Therefore, Sa 190 is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian erythrocyte micronucleus test.