Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: clastogenicity/aneugenicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-02-04 to 2013-03-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-(octylimino)bisethanol
EC Number:
239-555-0
EC Name:
2,2'-(octylimino)bisethanol
Cas Number:
15520-05-5
Molecular formula:
C12H27NO2
IUPAC Name:
2,2'-(octylimino)bisethanol

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: minimum 7 weeks
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: 5 animals of identical sex per cage, IVC cage (Polysulphone), Type II L
- Diet (ad libitum): Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): at least 10 x per hour
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: Cottonseed Oil;
- Justification for choice of solvent/vehicle: The solvent was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 10 mg/mL (1MTD), 5 mg/mL (0.5 MTD), 2 mg/mL (0.2 MTD)
- Amount of vehicle (if gavage or dermal): 10 mL per kg body weight
- Lot/batch no. (if required): MKBJ0602V (Sigma)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was diluted in Cottonseed Oil within 1 h before treatment. All animals received a single volume ip of 10 mL/kg bw.
Frequency of treatment:
The animals received the test item once ip.
Post exposure period:
Sampling of the peripheral blood was carried out on animals 44 (all controll and dose groups) and 68 h (negative control, 1MTD) after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
three dose groups: 1 MTD (100 mg/kg bw), 0.5 MTD (50 mg/kg bw), 0.2 MTD (20 mg/kg bw)
Basis:
other: diluted in Cottonseed Oil
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control: clastogenic control substance, good stability at room temperature, broad basis of historical laboratority data
- Route of administration: intraperitoneal
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
peripheral blood erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preceding study on toxicity was performed with the same strain and under identical conditions as in the main study. Three animals of each sex were treated ip for detection of the maximum tolerable dose. The maximum dose that was applied was 2000 mg/kg bw according the OECD 474 guideline. The maximum volume which was administered was 10 mL/kg bw. The highest dose group evaluated in the main experiment (100 mg/kg bw) based on the toxicity observed in the pre-experiment.


METHOD OF ANALYSIS:
Samples, including those of positive and negative controls, were evaluated using a flow cytometer (FACScan, BD Biosciences). Anti-CD71 antibodies were labelled with Fluorescein-isothiocyanate (FITC), anti-CD61 antibodies were labelled with Phycoerythrin (PE). Particles were differentiated using Forward Scatter (FSC) and Side Scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on the FL1, FL2 and FL3 channels for FITC, PE and PI respectively. At least 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect occurring possible cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The results were expressed as relative PCE (rel. PCE = proportion of polychromatic [immature] erythrocytes among total 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 OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion 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 was used. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated.
One male mouse received a single dose of 2000 mg/kg bw ip and died immediately after treatment. One male mouse received a single dose of 1000 mg/kg bw ip and died four minutes after treatment. One male mouse received a single dose of 200 mg/kg bw ip and died four minutes after treatment. One male mouse received a single dose of 150 mg/kg bw ip and died immediately after treatment. One female mouse received a single dose of 300 mg/kg bw ip and died three minutes after treatment. Three female mice received a single dose of 200 mg/kg bw ip. Two mice died immediately after treatment. The third mouse showed signs of toxicity such as reduction of spontaneous activity, bradykinesia, ataxia, opisthotonos, half eyelid closure, kyphosis and piloerection. One female mouse received a single dose of 150 mg/kg bw ip and showed signs of toxicity such as recumbency, constricted abdomen, reduction of spontaneous activity, opisthotonos, exophthalmos, bradykinesia and piloerection. Three male and three female mice received a single dose of 100 mg/kg bw ip. The male mice showed signs of toxicity such as reduction of spontaneous activity, constricted abdomen, recumbency, bradykinesia, opisthotonos, tremor, ataxia, catalepsis, piloerection, eye closure and half eyelid closure. The female mice showed signs of toxicity such as reduction of spontaneous activity, constricted abdomen, recumbency, ataxia, opisthotonos, bradykinesia, opisthotonos, piloerection, half eyelid closure, tremor and exophthalmos.

RESULTS OF DEFINITIVE STUDY
- Toxicity in the main experiment:
100 mg/kg bw was tested as the maximum tolerable dose (1 MTD) in the main experiment. The volume administered was 10 mL/kg bw ip.
All animals treated with the highest dose group (1 MTD) showed toxic effects. The animals treated with 50 mg/kg bw (0.5 MTD) and 20 mg/ kg bw (0.2 MTD) showed slight and/or no toxic effects after the treatment with the test item, respectively.

- Induction of micronuclei (for Micronucleus assay):
The negative controls (44 h and 68 h) evaluated were within the range of the historical control data of the negative control (0.08 – 0.34%). The mean values of micronuclei observed for the negative control (44 h) were 0.19% (male mice) and 0.23% (female mice). The mean values for the 68 h negative control were 0.19% (male mice) and 0.22% (female mice). The mean values of micronuclei observed after treatment with 0.2 MTD were 0.18% (male mice) and 0.22% (female mice). The values were within the range of the corresponding negative control and of the historical negative control data. Themean values noted for the 0.5 MTD dose group were 0.23% (male mice) and 0.21% (female mice). The values were within the range of the corresponding negative control and within the historical negative control data range. The dose group treated with 1 MTD (44 h treatment) showed mean values of 0.27% (male mice) and 0.24% (female mice). The values were within the range of the corresponding negative control and within the range of the historical negative control data. The mean values observed for the 1 MTD 68 h treatment were 0.28% (male mice) and 0.22% (female mice). The value observed in the male mouse was statistically significantly increased compared to the corresponding negative control. However, it 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 control and of the historical negative control data. As the increase observed in the 1 MTD 68h male (0.28%) was clearly within the range of the historical negative control data (0.08-0.34%) it can be stated, that no biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

- Ratio of PCE/NCE (for Micronucleus assay):
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 control data of the negative control (1.19 - 4.30). The mean values noted for the 44 h negative control were 2.20 (male mice) and 2.71 (female mice). The mean values detected for the 68 h negative control were 2.66 (male mice) and 3.20 (female mice).
The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.81 (male mice) and 1.74 (female mice). The mean values observed in the male and female groups were decreased compared to the corresponding negative control, but these decreases were not statistically significant.Moreover, the values were within the range of the historical control data.
The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 1.89 (male mice) and 1.29 (female mice). The mean values observed in the male and female groups were decreased compared to the corresponding negative control, but these decreases were not statisticallysignificant. Moreover, the values were within the range of the historical control data.
The animals who received 1 MTD (44 h treatment) showed mean values of 2.68 (male mice) and 1.84 (female mice). The value observed in the male group was increased compared to the corresponding negative control. The value observed in the female group was decreased compared to the corresponding negative control. However this increase/decrease was not statistically significant and the values were within the range of the historical control data.
The animal group which was treated with 1 MTD (68 h treatment) showed mean values of the relative PCE of 2.87 (male mice) and 2.48 (female mice). The value observed in the male group was slightly increased and in the female group decreased compared to the corresponding negative control. However this increase/decrease was not statistically significant and the values were within the range of the historical control data.

- Statistical evaluation:
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, except the value observed for the 1 MTD male group (68 hours). This group was significantly increased as compared to the corresponding control. However the value was within the range of the historical negative control data. Based on this data this increase was regarded as not biologically relevant.

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 did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

In a NMRI mouse peripheral blood micronucleus assay, five male and female animals per dose group were treated ip with the test item

at doses of 20, 50 and 100 mg/kg bw. Peripheral blood cells were harvested at 44h (all dose and control groups)and 68 h (negativ control and 1 MTD) post-treatment. The vehicle was Cottonseed Oil. The animals received the test item once ip. There were signs of toxicity during the study. the animals with doses of 0.2 and 0.5 MTD showed slight and/or no signs of systemic toxicity. The animals treated with 1 MTD showed signs of systemic toxicity such as reduction of spontaneous activity, constricted abdomen, half eyelid closure, recumbency, opisthotonos and tremor. The test item was tested at an adequate dose based on OECD 474. The positive control include the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time, except for the male animals treated with 1 MTD for 68 h. However, it was within the range of the historical negative control data. Therefore this increase was not regarded as biologically relevant. This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395, OECD 474 for Mammalian Erythrocyte Micronucleus Test.