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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 April 2019 - 21 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
9th Addendum to the OECD Guideline for Testing of Chemicals, Section 4, No. 474, adopted July 29, 2016, „Mammalian Erythrocyte Micronucleus Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), Part B: Methods for the determination of toxicity and other health effects: Mutagenicity – In vivo Mammalian Erythrocyte Micronucleus Test, No B.12; No L 142.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA 712-C-98-226, August 1998.
Version / remarks:
Environmental Protection Agency, Health Effects Test Guidelines OPPTS 870.5395 “Mammalian Erythrocyte Micronucleus Test“, EPA 712-C-98-226, August 1998.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
EC Number:
837-106-9
Cas Number:
71472-57-6
Molecular formula:
C9H11NO
IUPAC Name:
6-methyl-3,4-dihydro-2H-1,4-benzoxazine
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
Test system: Mice, NMRI
Rationale: Recognised as the recommended test system.
Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
Number of animals for the pre-test: 2 males and 2 females for each pre-test
Number of animals for the main study: 36 males
Age (beginning of treatment): 6 – 8 weeks
Body weight: 31.3(g) - 37.2(g)
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type II / III, with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 + 2°C
relative humidity approx. 45-65%
artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: On the day of the experiment, the test item was formulated in corn oil. The vehicle was chosen due to its relative non-toxicity for the animals and ability to form a suitable dosing formulation.
- Amount of vehicle (if gavage or dermal): All animals received a single volume of 10 mL/kg b.w. once orally by gastric gavage.
Details on exposure:
All animals received a single volume of 10 mL/kg b.w. once orally by gastric gavage.
The preparations were made freshly before the dosing occasion.
Duration of treatment / exposure:
24 - 48 hours
Frequency of treatment:
The animals received the test item, the vehicle or the positive control item once orally.
Post exposure period:
The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at time intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Vehicle
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Low dose
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Medium dose
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
High dose
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
Positive control
No. of animals per sex per dose:
Six males were treated per dose group and sampling time.
Control animals:
yes
Positive control(s):
Cyclophosphamide
Purity: 97% dissolved in sterile water.
Route and frequency of administration: orally once by gastric gavage
Concentration: 40 mg/kg b.w.
Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:
micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
Details of tissue and slide preparation:
Main Study Dose Selection
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly, or 2000 mg/kg b.w. as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes visually noticeable toxic reactions but not death or evidence of pain, suffering or distress necessitating humane euthanasia.
The administered volume was 10 mL /kg b.w. each treatment.
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken 48 h after treatment.

Treatment
At the beginning of the treatment and at the end of the in-life phase the animals (including the controls) were weighed and the individual dose volume was adjusted to the animal’s body weight. The animals received the test item, the vehicle or the positive control item once orally. Six males were treated per dose group and sampling time.
The animals of all dose groups, except the positive control group, were examined for acute toxic symptoms at time intervals of around 0-1 h, 2-4 h, 5-6 h, 24 h, and/or 48 h after administration of the test item. Sampling of the bone marrow was done 24 h after treatment for all treatment groups including the positive control, and at 48 h after treatment for the high dose group only.

Preparation of the Animals
The animals were sacrificed using CO2 followed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Analysis of Cells
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 4000 polychromatic erythrocytes (PCE) per animal were analysed for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per total erythrocytes. The analysis was performed with coded slides.
All animals per test group were evaluated as described.
Evaluation criteria:
A test substance is classified as positive in the assay if
a) 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,
b) This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
c) Any of these results are outside the distribution of the historical negative control data (e.g., Poisson-based 95% control limits).
There is no requirement for verification of a clearly positive or negative response. In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations.
A test substance that fails to produce a biologically relevant increase in the number of micronucleated polychromatic erythrocytes, applying above mentioned criteria, is considered negative in this system, given that there is evidence for bone marrow exposure (e.g., the substance can be detected in the blood by bioanalytical methods).
Statistical methods were used as an aid in evaluating the results.
Statistics:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test using the validated statistical program RScript Wilcoxon_2.Rnw. The Holm-Bonferroni Adjustment method was used to correct for the familywise error rate of the multiple comparisons.
A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.

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:
The test substance was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test substance was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w..
At time intervals of 24 h and 48 h after a single oral administration of the test item the bone marrow cells were collected for micronuclei analysis.
Six males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes (PCEs) were scored for micronuclei.
To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 500 erythrocytes.
The following dose levels of the test item were investigated:
24 h preparation interval: 50, 100, and 200 mg/kg b.w..
48 h preparation interval: 200 mg/kg b.w..
As estimated by two pre-experiments treatment at 200 mg BN-02 per kg b.w. (the maximum tolerated dose) was suitable as the highest treatment dose.
Clinical symptoms in the main experiment included partially closed eyes, piloerection, and decreased activity for all dose levels. Additionally, some animals of the mid and high dose showed discoloration of the urine (orange) and the symptoms closed eyes and lachrymation were observed in the high dose group only.
The observed systemic toxicity at the tested doses and the discoloration of the urine are indicative of a systemic distribution of the test item. Thus, bioavailability of the test item to the bone marrow under the tested conditions reported is assumed.
The mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control, indicating that the test substance did not have any cytotoxic properties in the bone marrow.
In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation time interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were below or near to the value of the vehicle control group and well within historical vehicle control values.
A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.
Treatment with 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

In the main experiment for each test item dose group 6 males received the test item dissolved in corn oil once orally. The volume administered was 10 mL/kg b.w.. The clinical symptoms observed following treatment are shown in the following table for each dose group, which indicates the number of animals with findings.

 

hours post-treatment (males)

Clinical symptoms

1

2-4

5-6

24

48

High dose: 200 mg/kg b.w. (12 males at 1 to 24 h; 6 males at 48 h)

Partially Closed Eyes

5

1

3

2

0

Closed Eyes

3

2

0

0

0

Piloerection

12

10

6

5

6

Decreased Activity

11

2

0

2

0

Stained Urine (orange)

0

0

7

0

0

Lachrymation

0

0

0

1

0

 

hours post-treatment (males)

Clinical symptoms

1

2-4

5-6

24

48

Medium dose: 100 mg/kg b.w. (6 males at 24 h)

Partially Closed Eyes

2

2

2

0

 

Piloerection

4

4

2

0

 

Decreased Activity

3

1

1

0

 

Stained Urine (orange)

0

0

3

0

 

Low dose: 50 mg/kg b.w.(6 males at 24 h)

Partially Closed Eyes

2

1

0

1

 

Piloerection

4

6

2

1

 

Decreased Activity

3

4

1

1

 

The animals treated with the vehicle control (corn oil) did not express any clinical symptoms.

 

Test
Group

Dose
mg/kg
b.w.

Sampling
time

Mean MN/4000 PCE

SD MN/4000 PCE

Range

Ratio
 PCE /total Ery

% ratio
Vehicle

min

max

Vehicle

0

24

4.8

2.8

2

10

0.632

100.00

Dose 1

50

24

3.0

1.1

2

4

0.637

100.79

Dose 2

100

24

7.3

4.5

3

13

0.629

99.53

Dose 3

200

24

6.5

4.1

1

12

0.626

99.05

Positive

40

24

114.7

60.5

36

216

0.634

100.32

Dose 3

200

48

3.7

2.7

0

7

0.576

91.14

MN = micronuclei

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this in-vivo micronucleus assay.
Executive summary:

This study was peformed to investigate the potential of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The dose volume administered orally was 10 mL/kg b.w. The administered volume of the positive control was 10 mL/kg.

At time intervals of 24 h and 48 h after a single oral administration of the test item bone marrow cells were collected for micronuclei analysis. Six males per test group were evaluated for the occurrence of micronuclei. Per animal 4000 polychromatic erythrocytes were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per total erythrocytes. The following dose levels of the test item were investigated in the main experiment:

24 h preparation interval: 50, 100, and 200 mg/kg b.w.
48 h preparation interval: 200 mg/kg b.w.

The highest dose (200 mg/kg b.w.) was estimated by two pre-experiments to be suitable.

Clinical symptoms in the main experiment included partially closed eyes, piloerection and decreased activity for all dose levels. Additionally, some animals of the mid and high dose showed discoloration of the urine (orange) and the symptoms closed eyes and lachrymation were observed in the high dose group only. The observed systemic toxicity at the tested doses and the discoloration of the urine are indicative of a systemic distribution of the test item. Thus, bioavailability of the test item to the bone marrow under the tested conditions reported is assumed. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test substance did not exert any cytotoxic effects in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronulclei at any preparation time interval after administration of the test item and with any dose level used.

A linear regression (least squares, calculated using the validated statistical program RScript LM_v02.Rnw) was performed to assess a possible dose dependent increase of mean micronuclei values. The mean number of micronuclei obtained for the groups treated with the test item was compared to the vehicle control group. A trend is judged as significant whenever the p-value (probability value) is below 0.05. A p-value of 0.4072 was obtained, demonstrating that there was a no dose dependent increase of mean micronuclei values.

Treatment with 40 mg/kg b.w. cyclosphosphamide administered orally was used as positive control which induced a substantial increase in cells with micronuclei.

Conclusion

In conclusion, in can be stated that under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the substance is considered to be non-mutagenic in this in-vivo micronucleus assay.

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