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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:
2017-10-17 to 2018-02-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2016
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian bone marrow erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Nonane-1,9-diol
EC Number:
223-517-5
EC Name:
Nonane-1,9-diol
Cas Number:
3937-56-2
Molecular formula:
C9H20O2
IUPAC Name:
nonane-1,9-diol
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Chemical name: 1,9-Nonanediol
- CAS no.: 3937-56-2
- Source and lot/batch No.of test material: Kuraray / 72732
- Expiration date of the lot/batch: 19 April 2018
- Molecular weight: 160.254 g/mol
- Purity: 99.23%

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
Test animals:
Species: Mouse
Strain: NMRI
Age: 6-10 wks
Weight at dosing: 32.4 - 37g
Source: Charles River, 97633 Sulzfeld, Germany
Acclimation period: 7 days
Diet: Altromin 1324 (Batch: 0426) maintenance diet for rats and mice, ad libitum
Water: Municipal water, ad libitum
Housing: Housed 5 animals of the same sex/cage

Environmental conditions
Temperature: 19-25°C
Humidity: 45-65%
Air changes: 10/hour
Photoperiod: 12 hours light/dark

Administration / exposure

Route of administration:
intravenous
Vehicle:
Cottonseed oil (dose volume: 10 mL/kg bw)
Details on exposure:
The test article was suspended in cottonseed oil and administered orally via gavage to three groups of male mice, 5/group at dose levels of 200, 500, 1000 mg/kg bw/d. the maximum dose administered was deemed to be a maximum tolerated dose based on a range finder experiment. Two further groups were included, one dosed with the vehicle control using the same dosing regimen as previously described and a further group dose with the positive control, cyclophosphamide (CPA). CPA (40 mg/kg bw) was administered as a single intraperitoneal injection 24 h prior to necropsy.
Duration of treatment / exposure:
Vehicle control and test article: dose orally via gavage at 0, 24 h
Postive control: dose intraperitoneally 24 h before necropsy
Frequency of treatment:
single dose as indicated above
Post exposure period:
24 h post final dose
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle (cottonseed oil)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
40 mg/kg bw (total dose)
Remarks:
Postive control (CPA)
No. of animals per sex per dose:
5 animals/group
Control animals:
yes
Positive control(s):
CPA (40 mg/kg bw) was administered as a single intraperitoneal injection 24 h prior to necropsy

Examinations

Tissues and cell types examined:
2000 immature erythrocytes/slide (4000/animal) were analysed for micronucleated immature erythrocytes.
Bone marrow toxicity was determined by counting the number of immature and mature erythrocytes (expressed as a ratio). The total number of erythrocytes counted/animal was 500.
Details of tissue and slide preparation:
Bilateral femurs from each mouse were injected with FBS to flush out bone marrow erythrocytes. Following centrifugation the pellet was resuspended in a small amount of FBS and 1 drop of the erythrocyte suspension was placed on to a microscope slide and smeared. Four slides were prepared/animal. After air drying, slides were fixed with methanol. Slides were coded and then stained with May-Grünwald/Giemsa.
Evaluation criteria:
Evaluation criteria:
Providing all acceptability criteria were fulfilled, a test item was considered clearly positive if:
- at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

If only the highest dose is examined at a particular sampling time, a test item is considered clearly positive if there is a statistically significant increase compared with the concurrent negative control and the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits)

Providing that all acceptability criteria are fulfilled, a test item was considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.
Statistics:
Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control group and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).

The Chi-squaresd test for trend at a statistical significance level of 5% (p <0.05, two-tailed) was used to test whether there was a dose-related increase in the micronucleated cells frequency of the dose groups of the 24 h sampling time.

Statistical methods were performed using the software GraphPad Prism version 6.0.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
A. Formulation analysis
Not applicable, not undertaken

B. Range finding test:
In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. One male and one female mouse received a single dose of 2000 mg/kg bw orally and showed very strong toxicity such as reduction of spontaneous activity, prone position, bradykinesia, ataxia, constricted abdomen, muscle spams (opisthotonos) and fast breathing. Based on animal welfare aspects both animals were euthanized some minutes after application.
The dose was reduced to 500 mg/kg bw and applied to one male and one female mouse twice at a 24 h interval. Both animals showed mild signs of systemic toxicity such as reduction of spontaneous activity, hunched posture, ataxia, bradykinesia, piloerection and half eyelid closure.
The dose was increased to 1000 mg/kg bw and dosed to three male and female mice twice at a 24 h interval. The animals showed moderate toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, bradykinesia, constricted abdomen, abnormal breathing, piloerection and half eyelid closure.

Due to the results obtained in the pre-experiment a dose of 1000 mg/kg bw/day was selected as the maximum tolerated dose (1 MTD). In the absence of substantial inter-sex differences in toxicity (a difference in MTD of 2-fold or greater), or likely sex-specific human exposure, only males were used for the main experiment

C. Micronucleus assay:
1. Observations: All animals survived to the scheduled necropsy.
200 mg/kg bw/d: toxicity limited to half eyelid closure 30 minutes after the first application.
500 mg/kg bw/d: mild signs of toxicity such as reduction of spontaneous activity, hunched posture and half eyelid closure up to 2 h after application.
1000 mg/kg bw/d: moderate toxic effects after both applications .Two hours post the final application no toxic symptoms were observed.
2. Bodyweight: No test article related effects were observed, generally all animals gained weight with the exception of 3 animals from the high dose group which had minimally lost weight (up to 2%) (refer to Table CA 7.6.2/01-1).
3. Immature erythrocyte ratio (toxicity) [PCE:NCE]: Refer to Table CA 7.6.2/01-2
The group mean negative control data was within the historical control limits of the negative control (95% confidence limit: 0.45 - 0.80). The mean value of the relative PCE noted for the negative control was 0.45.
200 mg/kg bw/d: relative PCE of 0.37. The value observed in this group was decreased compared to the concurrent negative control but this decrease was not statistically significant.
500 mg/kg bw/d relative PCE of 0.40. The value observed in this group was within the range of the concurrent negative control.
1000 mg/kg bw/d: relative PCE of 0.43. The value observed was within the range of the concurrent negative control.
A marginal dose related increase in group mean %PCE frequency was observed in treated animals compared to the concurrent vehicle control. Whilst this could be considered evidence of mild erythropoiesis, individual animal values were variable, no significant increase in %PCE was observed. Therefore it is concluded that the marginal increase in %PCE is considered unrelated to treatment and consistent to with the laboratory historical control range.
Animals treated with the positive control, CPA showed neither an increase (evidence of erythropoiesis) or decrease (evidence of bone marrow toxicity) in PCE ratio.
4. Micronucleated polychromatic erythrocytes (MN PCE): Refer to Table CA 7.6.2/01-2
All animals treated with 1,9-Nonanediol exhibited both group mean and individual MN PCE which were comparable with both the concurrent vehicle control group and the laboratory’s historical solvent control data. The incidence of MN PCE in the bone marrow of all negative control animals were within the historical solvent control data range (mean ±2 S.D.) and positive control animals gave the expected response.
The criteria for an acceptable assay were met and the assay was therefore considered sensitive.

D. Bioanalysis:
The test item 1,9-Nonanediol could be detected in plasma of one animal where blood was obtained 2 h after final application and in one animal where blood was obtained 3 h after the final application. For all other animals the quantification showed values below the lower limit of detection.
However, the detectability of the test item in blood plasma of the animals demonstrates systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.

E. Deficiencies
None.

Any other information on results incl. tables

Table CA 7.6.2/01-1:
Overview of bone marrow micronucleus study in mice treated orally via gavage with 1,9-Nonanediol: terminal bodyweight (g)

Parameter

Vehicle control: cottonseed oil

Animal no.

V1

V2

V3

V4

V5

Individual bwt

37.5

34.0

35.8

36.5

35.3

Group mean (±SD)

35.8 ±4.9

Parameter

Low dose group: 200 mg/kg bw/d

Animal no.

L1

L2

L3

L4

L5

Individual bwt

32.4

34.8

32.8

34.6

34.2

Group mean (±SD)

33.8 ±1.1

Parameter

Mid dose group: 500 mg/kg bw/d

Animal no.

M1

M2

M3

M4

M5

Individual bwt

37.2

34.1

36.4

32.6

33.6

Group mean (±SD)

34.8 ±1.9

Parameter

High dose group: 1000 mg/kg bw/d

Animal no.

H1

H2

H3

H4

H5

Individual bwt

33.4

36.8

35.9

36.1

36.6

Group mean (±SD)

35.8 ±1.4

Parameter

Positive control: CPA 40 mg/kg bw

Animal no.

P1

P2

P3

P4

P5

Individual bwt

33.2

36.9

33.8

35.4

37.0

Group mean (±SD)

35.3 ±1.7

Table CA 7.6.2/01-2:
Overview of bone marrow micronucleus study in mice treated orally via gavage with 1,9-Nonanediol: individual and group mean MN PCE and PCE:NCE

 

MN PCE/4000 PCE

%MN PCE

PCE:NCE

 

Parameter

Vehicle control: cottonseed oil

 

Animal no.

V1

V2

V3

V4

V5

V1

V2

V3

V4

V5

V1

V2

V3

V4

V5

 

Individual score

4

6

6

14

13

0.1

0.15

0.15

0.35

0.33

0.45

0.49

0.36

0.47

0.48

 

Group mean (±SD)

8.6 ±4.6

0.22 ±0.11

0.45 ±0.05

 

Parameter

Low dose group: 200 mg/kg bw/d

 

Animal no.

L1

L2

L3

L4

L5

L1

L2

L3

L4

L5

L1

L2

L3

L4

L5

 

Individual score

8

6

6

3

3

0.20

0.15

0.15

0.08

0.08

0.27

0.42

0.27

0.55

0.36

 

Group mean (±SD)

5.2 ±2.2

0.13 ±0.05

0.37 ±0.12

 

Parameter

Mid dose group: 500 mg/kg bw/d

 

Animal no.

M1

M2

M3

M4

M5

M1

M2

M3

M4

M5

M1

M2

M3

M4

M5

 

Individual score

8

10

6

10

14

0.20

0.25

0.15

0.25

0.35

0.47

0.40

0.36

0.38

0.37

 

Group mean (±SD)

9.6 ±3.0

0.24 ±0.07

0.40 ±0.05

 

Parameter

High dose group: 1000 mg/kg bw/d

 

Animal no.

H1

H2

H3

H4

H5

H1

H2

H3

H4

H5

H1

H2

H3

H4

H5

 

Individual score

4

7

5

25

14

0.10

0.18

0.13

0.63

0.35

0.48

0.41

0.42

0.45

0.39

 

Group mean (±SD)

11 ±8.7

0.28 ±0.22

0.43 ±0.04

 

Parameter

Positive control: CPA 40 mg/kg bw

 

Animal no.

P1

P2

P3

P4

P5

P1

P2

P3

P4

P5

P1

P2

P3

P4

P5

 

Individual score

92

157

81

100

153

2.30

3.92

2.03

2.50

3.83

0.44

0.45

0.40

0.46

0.45

 

Group mean (±SD)

117 ±35.7

2.92 ±0.89**

0.44 ±0.02

 

 

Historical control data (2012-2013)

 

 

Vehicle control

Positive control

 

PCE:NCE

%MN PCE

PCE:NCE

%MN PCE

 

 

No. of studies.:
Total animal no.:

6
30

6
30

 

Group mean

Mean ±S.D:
Range (min – max):
Control range (95%):

0.62 ±0.09
0.44 – 0.70
0.45 – 0.80

0.19 ±0.06
0.12 – 0.27
0.07 – 0.31

0.64 ±0.05
0.53 – 0.69
0.53 – 0.74

2.36 ±0.78
1.31 – 3.50
0.81 – 3.91

 

Individual values

Mean ±S.D:
Range (min – max):
Control range (95%):

0.62 ±0.14
0.31 – 0.90
0.34 – 0.90

0.19 ±0.12
0.00 – 0.50
0.00 – 0.43

0.64 ±0.13
0.37 – 0.91
0.38 – 0.89

2.36 ±1.21
0.40 – 4.65
0.00 – 4.48

 

**p= 0.01 (p 0.0079)

Trend test: Not significant (p 0.1491)

PCE:NCE: polychromatic erythrocyte : normochromatic erythrocyte

MN PCE: Micronucelated polychromatic erythrocyte

CPA: cyclophosphamide

 

Applicant's summary and conclusion

Conclusions:
It can be concluded that 1,9-Nonanediol did not show an increase in the incidence of micronuclei in male mouse bone marrow polychromatic erythrocytes following dosing via oral gavage up to a level of 1000 mg/kg bw/day (a dose deemed to be a maximum tolerated dose) under the experimental conditions described. Bioanalysis data confirmed test article exposure to the target organ.
Executive summary:

In the preliminary dose range finding study male mice were dose via oral gavage of1,9-Nonanediol suspended in cottonseed oilvehicle at either 2000, 1000 and 500 mg/kg bw/d (employing a dose volume of 10 mL/kg). Mice received a dual dose of the test article formulation, with a 24 hour period between doses.Mice dosed at 2000 mg/kg bw/day showed very strong toxicity such as reduction of spontaneous activity, prone position, bradykinesia, ataxia, constricted abdomen, muscle spasms and fast breathing. Based on animal welfare aspects both animals were euthanized some minutes after application. The dose was reduced to 500 mg/kg bw/d and applied to 1 male and 1 female mice twice. Both animals showed mild signs of systemic toxicity such as reduction of spontaneous activity, hunched posture, ataxia, bradykinesia, piloerection and half eyelid closure.

The dose was increased to 1000 mg/kg bw and dosed to 3 male and 3 mice. The animals showed moderate toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, bradykinesia, constricted abdomen, abnormal breathing, piloerection and half eyelid closure.The maximum tolerated dose was deemed to be 1000 mg/kg bw/day.

In the micronucleus experiment groups of 5 male mice were dosed with vehicle (cottonseed oil), 1,9 -Nonanediol (200, 500, 1000mg/kg bw/d) using the same dosing regimen, dose volume and route as previously stated. A positive control (cyclophosphamide, CPA: 40 mg/kg bw) was included, with mice receiving a single dose via intraperitoneal injection (dose volume 10 mL/kg) on day 2. All mice were killed 24 hours post the final dose, bone marrow harvested and slides prepared for micronucleated polychromatic erythrocyte (MN PCE) and polychromatic erythrocyte (PCE) : normochromatic erythrocyte (NCE) frequency were analysed on 5 mice/group.

For analytical purposes blood samples of additional two animals/time point were obtained two hours, three hours and four hours after the final application of the maximum tolerated dose of the test item that were stored at -80°C as plasma sample

All animals treated with1,9-Nonanediolexhibited both group mean and individual MN PCE which were comparable with both the concurrent vehicle control and the laboratory’s historical solvent control data. All animals treated with the positive control exhibited marked increases in MN PCE such that the frequency of MN PCE in the positive control group was significantly (p< 0.01) greater than the observed frequency in the concurrent vehicle control group. The frequency of MN PCE in the concurrent negative control group was within the distribution (mean ±2SD) of laboratory’s historical solvent control data. The criteria for an acceptable assay were met and the assay was therefore considered sensitive.

The groups that were treated with 1,9-Nonanediolshowed no test article related increase or decrease in the ratio of PCE:NCE compared to the concurrent vehicle control group. The positive control group showed neither an increase (evidence of erythropoiesis) or decrease (evidence of bone marrow toxicity) in PCE ratio.

The test item 1,9-Nonanediol could be detected in plasma of one animal where blood was obtained 2 h after final application and in one animal where blood was obtained 3 h after the final application. For all other animals the quantification showed values below the lower limit of detection. However, the detectability of the test item in blood plasma of the animals demonstrates systemic bioavailability after oral administration which is considered as evidence for exposure of bone marrow to the test item.

It can be concluded that1,9-Nonanedioldid not show an increase in the incidence ofmicronuclei in male mouse bone marrow polychromatic erythrocytesfollowing dosing via oral gavage up to a level of 1000 mg/kg bw/day (a dose deemed to be a maximum tolerated dose) under the experimental conditions described. Bioanalysis data confirmed test article exposure to the target organ.