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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 23 November 2016 Experimental completion date 02 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
liquid
Details on test material:
Name: PREPOLYMER D
Chemical Name: Reaction mass of Bis (2-chloroethoxy)methane and 1,15-dichloro-3,5,8,11,13-pentaoxa-pentadecane and 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane
Batch/Lot Number:1603101
Purity: 100% (multi constituent substance)
Appearance: Extremely pale yellow liquid
Specific details on test material used for the study:
Identification: Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4
Physical state/Appearance: Extremely pale yellow liquid
Batch: 1603101
Purity: 100%
Expiry Date: 19 June 2017
Storage Conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 186.8 to 216.1 g, and were approximately 7 to 12 weeks old. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
A range-finding test was performed to find suitable dose levels of the test item following a triple oral administration at approximately 0, 24 and 44 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. Based on toxicology information supplied by the Sponsor the initial dose level was limited to 200 mg/kg. Additionally bone marrow slides were prepared and scored for PCE/NCE ratio as an indicator of toxicity to bone marrow.
Duration of treatment / exposure:
44 hours
Frequency of treatment:
Daily
Post exposure period:
Animals were observed approximately 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 18-24
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 25-31
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 32-38
No. of animals per sex per dose:
7 males rats per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Dose level Concentration Dose volume Kill time Animal numbers
(mg/kg) (mg/mL) (mL/kg) (hours after
initial dosing)
Positive control
(Cyclophosphamide) 25 2.5 10 24‡ 8 - 12

Positive Control
(N-Nitroso-N-methylurea) 25 2.5 10 24‡ 13 -17

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Micronucleus Slide Preparation
A 15 mL centrifuge tube was filled with approximately 2-3 mL of foetal bovine serum (FBS) and, using a suitable hypodermic needle mounted on a 2 mL syringe, a portion of the serum was withdrawn from the centrifuge tube into the syringe. The needle was pushed a few millimeters into the bone marrow canal and the marrow from 1 femur was flushed into the serum in the centrifuge tube.
Approximately 1 mL of the bone marrow suspension was transferred to a microfuge tube and centrifuged at approximately 200 g and the supernatant was removed using a Pasteur pipette leaving sufficient serum to just cover the top of the cell pellet. The cells were mixed to give a homogenous suspension. A small drop of each bone marrow suspension was transferred to the frosted end of four glass microscope slides and smears were made and air-dried. Two slides were stained for scoring of micronuclei and two reserved as spares.
The smears were fixed in absolute methanol for 20 minutes and stained with May Grünwald stain and then counterstained with Giemsa stain and subsequently rinsed with pH 6.8 buffer solution for 3 minutes and then rinsed in distilled water. After air drying the smears had a cover slip applied using a mounting medium.
Evaluation criteria:
Micronucleus Scoring
The stained slides were "blind" coded and examined using light microscopy at x 1000 magnification. 4000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei.
In addition, the number of normochromatic erythrocytes within a total of at least 1000 erythrocytes were counted and these cells were also scored for the incidence of micronuclei. The ratio of polychromatic/normochromatic erythrocytes (PCE/NCE), group mean values and standard deviations were calculated.
Statistics:
For the Micronucleus Test statistical analysis of MN/4000 PCE and PCE/NCE ratio data was performed where necessary using a t test following a  (x+1) transformation of the data.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Please see "Any other information on results incl. tables" for a summary of the group mean data for the Micronucleus Test

Any other information on results incl. tables

                         Micronucleus Test – Summary of Group Mean Data

TREATMENT GROUP

NUMBER OF PCE WITH MICRONUCLEI PER 4000 PCE

PCE/NCE RATIO

GROUP MEAN

SD

GROUP MEAN

SD

1.  Vehicle Control

2.4

1.6

1.34

0.49

      48-hour sampling time

2.   Positive Control

34.0*

22.2

0.67**

0.10

      24-hour sampling time

3.   List No 940-783

3.6

2.1

0.98

0.54

      200 mg/kg

      48-hour sampling time

4.   List No 940-783

3.6

2.4

1.36

0.72

      100 mg/kg

      48-hour sampling time

5.   List No 940-783

2.7

1.3

1.29

0.74

      50 mg/kg

      48-hour sampling time

PCENCESD******



PCE      =Polychromatic erythrocytes

NCE     =Normochromatic erythrocytes

SD       =Standard deviation

*          = P < 0.05

**        =P <0.01

 

Range-Finding Toxicity Test

The mortality data are summarized as follows:

Dose Level
(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

200

Male

2

oral

0

0

0

0/2

240

Male

2

oral

0

0

2

2/2

200

Male

2

oral

0

0

0

0/2

 

The following clinical signs were observed in the animals dosed with the test item at 200 mg/kg via the oral route: hunched posture, lethargy, ataxia, splayed gait and diuresis. The dose level was increased to 240 mg/kg and the following clinical signs were observed, hunched posture and lethargy. Both animals were found dead at the 48-hour observation time, indicating that lethal toxicity occurred after the third administration of test item at 240 mg/kg. A confirmatory range-finding test was performed on two animals at 200 mg/kg and the observations were considered to be relatively moderate (hunched posture and ataxia), confirming this was an acceptable maximum dose level to use in the main test.

Bone marrow slides were prepared for quantitative assessment from the three range-finding experiments. The slides were scored per 1000 cells and the data is presented below.  

Animal Code

Dose level (mg/kg)

Number of polychromatic erythrocytes

Number of Normochromatic erythrocytes

PCE/NCE ratio

1-0

200

460

540

0.85

1-1

351

649

0.54

2-0

240

290

710

0.41

2-1

254

746

0.34

3-0

200

417

583

0.72

3-1

348

652

0.53

 

The quantitative assessment revealed that bone marrow toxicity had occurred at both dose levels. The bone marrow toxicity that occurred at 200 mg/kg was considered to be moderate whilst at 240mg/kg was considered to be too marked.

Based on the above data the maximum tolerated dose (MTD) of the test item, 200 mg/kg, was selected for use in the main test, with 100 and 50 mg/kg as the lower dose levels. 

Micronucleus Main Test

Mortality Data and Clinical Observations

One premature death was observed in the 200 mg/kg (MTD) dose group. A second animal in the 200 mg/kg group died just prior to sampling and although it was processed for comet and micronucleus evaluation it was subsequently decided that any data would be compromised and it was therefore excluded from analysis. 

 

The following clinical signs were observed in animals dosed with test item at 200 mg/kg: hunched posture, ataxia, pilo erection, prostration, increased respiration, gasping respiration, labored respiration, ptosis and splayed gait. The more severe observations were most apparent at the 48 hour observation, approximately 4-hours after the last dose.

 Evaluation of Micronucleus Slides

Dose-related, decreases in the PCE/NCE ratio were observed in the 200 mg/kg test item dose groups when compared to the vehicle control group. These decreases, together with the observation of clinical signs, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Applicant's summary and conclusion

Conclusions:
In the Micronucleus element of the test, the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non-genotoxic under the conditions of the test.
Executive summary:

Summary

Introduction

The Micronucleus Assay is a standard follow-upin vivo study. 

The micronucleus test is a mammalianin vivotest which detects chemical-induced damage to the chromosomes or the mitotic apparatus. Polychromatic erythrocytes in the bone marrow of rats are used in this study. When the erythroblast develops into an erythrocyte, the main nucleus is extruded and may leave micronuclei in the cytoplasm. Observation of micronuclei is facilitated in these cells because they lack a nucleus. As micronuclei occur under normal conditions, this study is based on detecting an increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow of treated animals. 

Bone Marrow was sampled for micronucleus slides.

Methods

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 200 mg/kg with 100 mg/kg and 50 mg/kg as the lower dose levels. Groups, each of seven rats, were dosed three times at 0, 24 and 44 hours with the test item. Animals were killed 4 hours after the third dose administration. 

In addition, three further groups of rats were included in the study; one group (seven rats) was dosed via the oral route with the vehicle alone (arachis oil) at 0, 24 and 44 hours, and a second group (five rats) was dosed orally with cyclophosphamide (CP), once only, at the 24-hour time-point, to act as a positive control for the micronucleus element of the study. The third group of rats was dosed with N-Nitroso-N-methylurea (NMU) at 24 and 44-hours to act as the positive control for the Comet element of the study. 

 

The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 48 hours after the start of the test. The glandular stomach, duodenum, liver, bone marrow and testes were processed for comet slides. Samples of liver, duodenum, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. Comet slides were not prepared for the positive control animals dosed with CP.

Bone marrow was then extracted from the second femur, and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Micronucleus slides were not prepared for the positive control animals dosed with MNU.

Results

One premature death was observed in the 200 mg/kg (MTD) dose group. A second animal in this 200 mg/kg group died just prior to sampling and although it was processed for comet and micronucleus evaluation it was subsequently decided that any data may be compromised and this animal was therefore excluded from the final analysis. The following clinical signs were observed in animals dosed with test item at 200 mg/kg: hunched posture, ataxia, pilo erection, prostration, increased respiration, gasping respiration, labored respiration, ptosis and splayed gait. The more severe observations were most apparent at the 48 hour observation, approximately 4-hours after the last dose.

 

In the micronucleus test, dose-related, decreases in the PCE/NCE ratio were observed in the 200 mg/kg test item dose group when compared to the vehicle control group. Although not statistically significant, these decreases, together with the observation of clinical signs, were taken to indicate that systemic absorption had occurred and exposure to the target tissues had been achieved.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

 

The positive control group (cyclophosphamide) showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.


 

Conclusion

In the Micronucleus Test the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non genotoxic under the conditions of the test.