Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test (OECD TG 471) with BMI found no mutagenic activity under the test conditions employed.

A Chromosome Aberration test (OECD TG 473) conducted in Chinese Hamster Ovary (CHO) cells indicated clastogenic activity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 February 1988 - 18 April 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study report does not cite specific official test guidelines, however the report is thorough and the test conditions are well documented. It may be determined that the methodology is broadly consistent with modern test methods and the report does include a claim that the study was conducted according to the General Principles of Good Laboratory Practice; on this basis the study is considered reliable with restrictions.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Liver microsomal enzyme fraction
Test concentrations with justification for top dose:
2.5, 5, 10 and 20 μg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 24 hours (without S-9) and 6 hours (with S-9) + 18 hours in treatment free media

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (colcemid 0.1μg/mL) 2 hours before required harvest time
STAIN (for cytogenetic assays): 2% Gurrs Giemsa R66

NUMBER OF REPLICATIONS: 2 per treatment with S-9 and 2 without S-9

NUMBER OF CELLS EVALUATED: 100 consecutive well spread metaphases from each culture

DETERMINATION OF CYTOTOXICITY
- Method: count of cells % of control

COUNT OF CELLS with POLYPLOIDY: yes
Evaluation criteria:
KUEMS Guidelines for mutagenicity Testing (1983)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: CHO-K1 BH4cell line
Remarks:
Migrated from field 'Test system'.

Cytotoxicity

Cell counts for the treatment without S-9 24 hour treatment

 

Without S-9

Culture A

Culture B

Mean %

Concentration of MB-3000 (μg/mL)

No of cells

X105

% of control

No of cells

X105

% of control

0

3.75

100

2.86

100

100

2.5

2.96

79

3.06

107

93

5.0

2.35

63

2.69

94

78.5

10.0

1.86

50

1.69

59

54.5

20.0

1.34

36

1.59

56

46.0

EMS 1 mg/mL

1.84

49

1.78

62

55.5

Cell counts for the treatment with S-9 6 hour treatment

 

With S-9

Culture A

Culture B

Mean %

Concentration of MB-3000 (μg/mL)

No of cells

X105

% of control

No of cells

X105

% of control

0

1.80

100

1.52

100

100

2.5

1.91

106

1.45

95

100.5

5.0

1.66

92

1.61

106

99

10.0

1.23

68

1.37

90

79

20.0

1.07

59

0.68

45

52

CP 25 ug/mL

1.69

94

1.63

107

100.5

Result of Chromosome aberration assay

Treatment without S-9

Treatment group

Treatment time

(hr)

Con-centration

No of diploid cells scored

Cells with polyploidy

No of cells with observed structural aberrations

N

%

Judge-ment

GAPS

Chromatid

Chromosome

Others

Total No Cells with aberration

Judge-ment

g

ctb

cte

csb

cse

X

Z

-g

+g

Solvent control

24

0

100

10

(9.1)

-

1

0

0

0

0

0

0

2

3

-

100

11

(9.9)

2

0

0

0

0

0

0

0

2

200

21

(9.5)

3 (1.5)

0(0)

0(0)

2(1)

0(0)

0(0)

0(0)

2(1)

5(2.5)

MB-3000

24

2.5

100

12

(10.7)

-

1

2

0

2

1

0

0

5

6

+

100

8

(7.4)

5

0

0

4

1

0

0

5

10

200

20

(9.1)

6 (3)

2 (1)

0(0)

6(3)

2(1)

0(0)

0(0)

10(5)

16 (8)

5.0

100

17

(14.5)

-

4

0

0

1

0

0

0

1

5

+

100

6

(5.7)

4

0

0

5

0

2

1

7

10

200

23

(10.3)

8 (4)

0(0)

0(0)

6(3)

0(0)

2(1)

1(0.5)

8(4)

15(7.5)

10.0

100

11

(9.9)

-

7

0

1

9

0

0

1

10

15

+

100

8

(7.4)

4

4

1

4

1

0

6

8

12

200

19

(8.7)

11 (5.5)

4(2)

2(1)

13(6.5)

1(0.5)

0(0)

7(3.5)

18(9)

27(13.5)

20.0

100

9

(8.3)

-

3

5

2

5

2

0

4

13

15

+

100

9

(8.3)

6

2

1

5

0

1

5

7

13

200

18

(8.3)

9 (4.5)

7(3.5)

3(1.5)

10(5)

2(1)

1(0.5)

9(10.5)

20

28(14)

EMS

24

1000

50

6

(10.7)

-

22

17

21

14

3

4

0

43

44

-

50

3

(5.7)

14

12

15

19

3

3

0

33

39

100

9

(8.3)

36 (36)

29(29)

36 (36)

33(33)

6(6)

7(7)

0(0)

76(76)

83(83)

 

Treatment with S-9

Treatment group

Treatment time

(hr)

Con-centration

No of diploid cells scored

Cells with polyploidy

No of cells with observed structural aberrations

N

%

Judge-ment

GAPS

Chromatid

Chromosome

Others

Total No Cells with aberration

Judge-ment

g

ctb

cte

csb

cse

X

Z

-g

+g

Solvent control

6

0

100

14

(12.2)

-

6

1

1

3

0

0

0

5

11

-

100

2

(2.0)

4

1

1

3

1

0

0

6

10

200

16

(7.4)

10 (5)

2(1)

2(1)

6(3)

1(0.5)

0(0)

0(0)

11(5.5)

21(10.5)

MB-3000

6

2.5

100

10

(9.1)

-

1

0

0

1

1

0

0

2

3

+

100

8

(7.4)

1

0

0

0

0

0

0

0

1

200

18

(8.3)

2 (1)

0(0)

0(0)

1(0.5)

1(0.5)

0(0)

0(0)

2(1)

4 (2)

5.0

100

6

(5.7)

-

2

0

2

1

0

0

2

3

5

+

100

6

(5.7)

6

1

1

0

0

0

0

1

6

200

12

(5.7)

8 (4)

1(0.5)

3(1.5)

1(0.5)

0(0)

0(0)

2(1)

4(2)

11(5.5)

10.0

100

11

(9.9)

-

7

6

3

2

0

0

4

11

16

+

100

8

(7.4)

3

2

4

5

1

0

2

9

11

200

19

(8.7)

10 (5)

8(4)

7(3.5)

7(3.5)

1(0.5)

0(0)

6(3)

20(10)

27(13.5)

20.0

100

4

(3.8)

-

7

8

2

4

0

0

8

11

17

+

100

6

(5.7)

6

3

7

4

2

0

9

15

19

200

10

(4.8)

13 (6.5)

11(3.5)

9(4.5)

8(4)

2(1)

0(0)

17(8.5)

26(13)

36(18)

CP

6

25

50

8

(13.8)

-

9

9

14

29

2

0

0

38

40

-

50

8

(13.8)

8

11

7

33

1

0

0

42

43

100

16

(13.8)

17 (17)

20(20)

21(21)

62(62)

3(3)

0(0)

0(0)

80(80)

83(83)

 

Conclusions:
It was concluded that the test substance showed evidence of cytogenic and clastogenic activities in this in vitro mamalian cell system under the test conditions employed.
Executive summary:

A test was conducted (Safe Pharm Laboratories, 1988) in vitro in Chinese Hamster Ovary cells to assess the clastogenic potential of BMI in mammalian cells. The test was conducted using a 6-hour exposure period in the presence of metabolic activation (achieved by adding induced rat liver homogenate metabolising system) and a 24-hour exposure period in the absence of metabolic activation.

Significant dose-related increases in both toxicity and the frequency of aberrations were observed under both regimes of treatment with the test material. BMI was concluded to be clastogenic to CHO cells in vitro.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 16 July to 03 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Official notice of MHLW, METI and MOE, Yakushokuhatsu 0331 No 7; Seikyoku No 5; Kanpokihatsu No 110331009
Version / remarks:
31 March 2011
Qualifier:
according to
Guideline:
other: Notice of J MOL
Version / remarks:
8 February 1999
Qualifier:
according to
Guideline:
other: Japanese Ministry of Health and Welfate. Evaluation and Licensing Division, Pharmaceutical and Medical Safety Bureau, Notificiation No. 1604
Version / remarks:
1 November 1999
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-mix; S9 prepared from male rats induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Test 1: 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Test 2: 1.5, 5, 15, 50, 150 and 500 μg/plate in the absence of S9 mix; 5, 15, 50, 150, 500 and 1500 μg/plate in the presence of S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test substance was assessed at 50 mg/mL in water and in dimethyl sulphoxide (DMSO). It was insoluble in water, but was soluble in DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
In the absence of S9 mix for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
In the absence of S9 mix for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
In the absence of S9 mix for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
In the absence of S9 mix for strain WP2 uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
In the presence of S9 mix for strains TA100, TA1535 and WP2 uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
In the presence of S9 mix for strains TA98 and TA1537
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar

DURATION
First test (plate incorporation)
- Exposure duration: ca. 72 hours at 37 °C

Second test (pre-incubation method)
- Pre-incubation: 30 minutes at 37 °C
- Exposure duration: ca. 72 hours at 37 °C

NUMBER OF REPLICATIONS: 3

OTHER: After exposure period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter.

Evaluation criteria:
If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system. No statistical analysis is performed.
If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance should always be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Statistics:
None stated
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
below 500 μg/plate in the absence of S9 mix, and below 1500 μg/plate in the presence of S9 mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
below 500 μg/plate in the absence of S9 mix, and below 1500 μg/plate in the presence of S9 mix
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
First test
Toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance at concentrations of 500 μg/plate and above in the absence of S9 mix, and 1500 μg/plate and above in the presence of S9 mix. Precipitate was observed on all plates containing the test substance at concentrations of 500 μg/plate and above. Maximum exposure concentrations of 500 μg/plate in the absence of S9 mix and 1500 μg/plate in the presence of S9 mix were, therefore, selected for use in the second test.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration tested in either the presence or absence of S9 mix.

Second test
Toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was seen in all strains following exposure to the test substance at 500 μg/plate in the absence of S9 mix, and 1500 μg/plate in the presence of S9 mix. Precipitate was observed on all plates containing the test substance at concentrations of 500 μg/plate and above.
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration tested in either the presence or absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Conclusions:
It was concluded that BMI showed no evidence of mutagenic activity in this bacterial system under the test conditions employed.
Executive summary:

BMI was assessed for the ability to cause gene mutation inSalmonella typhimurium and Escherichia coli. The study was conducted in accordance with GLP and to OECD guideline 471 (Huntingdon Life Sciences, 2012). In this main study 4 strains ofSalmonella Typhimuriumand one strain of Escherichia Coli were treated with test substance in the absence and in the presence of S9 mix. For the first test, concentrations tested were up to 5000 µg per plate. Results were negative for all strains at all concentrations therefore a second test was conducted. This employed the pre-incubation assay. Maximum concentrations were 500 µg/plate in the absence of S9 mix and 1500 µg/plate in the presence of S9 mix. Again results were negative for all strains at all concentrations. Therefore there was no evidence of mutagenic activity in this bacterial system under the test conditions employed.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo Alkaline Comet Assay (OECD TG 489) in rats showed no indication of DNA strand breakage in the liver, glandular stomach or duodenum.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo alkaline comet (single cell get electrophoresis) assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 April 2018 - 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Eight to ten weeks
- Weight at study initiation: 188.0 to 218.1 g
- Assigned to test groups randomly: yes
- Fasting period before study: No
- Housing: Up to five animals per sex in solid-floor polypropylene cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25°C
- Humidity: 30 to 70%
- Air changes: Approximately 15 air changes per hour
- Photoperiod: 12 hours light / 12 hours dark

IN-LIFE DATES: From: 04 April 2018 To: 18 May 2018
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used:arachis oil
- Concentration of test material in vehicle: 50 - 200 mg/mL
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within 2 hours of it being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.
Duration of treatment / exposure:
Approximately 28 hours (two doses approximately 24 hours apart; animals were killed 4 hours after the second dose)
Frequency of treatment:
Two doses over a 24 hour period
Post exposure period:
4 hours
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Maximum recommended dose
No. of animals per sex per dose:
2 males and 2 females - range finding group
5 males only - 500 and 1000 mg/kg
7 males - 2000 mg/kg
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-methylurea
- Justification for choice of positive control: Substance has been found in-house to induce strand breaks and damage to DNA under the conditions of the test
- Route of administration: Oral gavage
- Doses / concentrations: Three male rats received two administrations of 25 mg/kg (10 mL/kg of a 2.5 mg/mL solution in water) approximately 24 hours apart
Tissues and cell types examined:
Liver, glandular stomach, duodenum
Details of tissue and slide preparation:
Tissue Sample Requirements
Humane euthanasia was performed on the animals at the end of the exposure period using a method that did not affect the integrity of the required tissues (carbon dioxide asphyxiation).
Samples of liver, glandular stomach, and duodenum were obtained from each animal for comet processing.

Sub-samples of the liver, glandular stomach and duodenum were taken from the vehicle control animals and the dose group animals and preserved in 10% buffered formalin for possible histopathology investigations. Assessment of cytotoxicity by histopathology may have been conducted if the results from the Comet assay, or other observations, suggest cytotoxicity may have been confounding the interpretation of the Comet assay.

The tissue samples were processed under subdued lighting and over ice to provide single cell suspensions, providing sufficient cells for scoring for the comet assay as follows:

Liver - A small piece of liver (approximately 1 cm3) was washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

Glandular Stomach – A small section of the glandular stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for
approximately 15 minutes on ice. The mucosal layer of the glandular stomach was removed by gentle scraping and then a single cell suspension was obtained by scraping the remaining
tissue into a small volume of stomach buffer.

Duodenum - Approximately a 2 cm piece of Duodenum was processed. This was immersed briefly in liver buffer and then scraped gently to remove any contents, incubated in liver
buffer (approximately 10 mL), on ice for 5 to 10 minutes. A single cell suspension was obtained by gentle scraping into approximately 1 mL of fresh liver buffer.


Slide Preparation:
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature. The slides were labelled for animal number, study number and tissue type prior to use for the comet assay.

Approximately 30 μL of the cell suspension was added to 270 μL of molten 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 μL of this agarose/cell suspension mix was placed onto a pre-coated slide. Two gels were placed on each slide, and 4 gels were prepared for each tissue. Two of the gels were scored for Comets (A and B replicates) and two (C and D replicates) were kept in reserve in case further scoring was required or the gels were damaged during processing. The agarose/cell suspension mix was immediately covered with a glass cover slip, transferred to a cold room at approximately 4 °C in the dark for approximately 20 minutes to allow it to solidify.

Once the LMP agarose had set, the cover slips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight. All slides went through the subsequent processing.
Following lysis, the slides were removed from the solution, briefly rinsed with neutralization buffer and placed onto the platform of an electrophoresis bath, which was filled with chilled electrophoresis buffer (pH>13), until the slide surface was just covered. The slides were then left for 20 minutes to allow the DNA to unwind, after which they were subjected to electrophoresis at approximately 0.7 V/cm (calculated between the electrodes), 300 mA for 20 minutes. The buffer in the bath was chilled during the electrophoresis period and the temperature of the electrophoresis buffer was monitored at the start of unwinding, the start of electrophoresis and the end of electrophoresis to ensure the electrophoresis solution was maintained at low temperature (2-10°C).

At the end of the electrophoresis period, the bath was switched off, the slides gently removed and placed on to a draining surface and drop wise coated with a neutralization buffer and left for at least 5 minutes. The slides were then drained and a repeat of the addition of the neutralization buffer was performed twice. The slides were further drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry.

Once dry the slides were stored prior to scoring. Two of the four processed slides were scored and the remaining slides were stored as backup slides.
Evaluation criteria:
The slides were stained just prior to analysis for comets. To each dry slide, 75 μL of propidium iodide (20 μg/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, Comet IV.

Two slides for each tissue per animal were scored with a maximum of 75 cells per slide giving an accumulative total of 150 cells per tissue per animal. Care was taken to guarantee that a cell was not scored twice. The slide score data for each tissue was processed using the Excel macro program provided in Comet IV version 4.3.1. Comparisons between the vehicle control group response and that of the test item dose groups was made. The primary endpoint was percentage DNA in the tail (percentage Tail intensity and median percentage Tail intensity).

Each slide was also assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity.
Statistics:
A comparison was made between the vehicle control groups and the positive control groups. The individual slide score data for the percentage tail intensity and median percentage tail intensity was compared using a Students t-test with a √1+x transformation. Comparisons between the vehicle control groups and the test item dose groups were also made when it was considered that there was a marked increase over the vehicle control value.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg, 2 males and 2 females
In animals dosed with test item there were no premature deaths and no clinical signs were observed.
Bone marrow slides were prepared from the range-finding experiments for quantitative assessment.
The quantitative assessment revealed that there was very modest bone marrow toxicity observed in one male and one female at 2000mg/kg which was considered to give an indication that systematic absorption of the test item had occurred.
Based on the above data the maximum tolerated dose (MTD) of the test item, 2000 mg/kg, was selected for use in the main test, with 1000 and 500 mg/kg as the lower dose levels. There was no noticeable difference in clinical signs between the male and female animals and therefore only male animals were used for the main test.

RESULTS OF DEFINITIVE STUDY
One animal from the 1000 mg/kg dose group was killed in extremis after the initial dose but this death was considered to be due to a technical error and not test item related. No clinical signs were observed in animals dosed with the test item, other than the animal which was killed prematurely. The loss of one animal from the 1000 mg/kg dose group is considered to have no impact on the study outcome since there were sufficient animals in the MTD group (2000 mg/kg) to meet the requirements of the guideline.

There were no statistically significant increases in percentage tail intensity for any of the test item dose levels in the liver, glandular stomach or duodenum tissues which exceeded the current historical control range for a vehicle, confirming the test item did not induce DNA damage in the liver, glandular stomach or duodenum. One animal in the 2000 mg/kg dose group had very high tail intensities in the glandular stomach compared with the rest of the group but this was considered to be an exception for one animal and although it increased the overall tail intensity for the group the result was not statistically significant.

There were no marked increases in hedgehog frequency for any of the test item dose levels in either of the tissues investigated. High numbers of hedgehogs observed in the glandular stomach and duodenum are characteristic of the gastro-intestinal tract where there is a high turnover of cells.
Conclusions:
The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity in the liver, glandular stomach or duodenum and therefore the test item was considered to be unable to induce DNA strand breakage to these tissues in vivo under the conditions of the test.
Executive summary:

As the available in-vitro data indicated the possibility of a mutagenic effect, an in-vivo Comet assay was conducted (Envigo, 2018) according to OECD TG 489 and in accordance with GLP principles. Three groups of male rats received two doses of BMI (500, 1000 and 2000 mg/kg) approximately 24 hours apart. Following dosing the rats were humanely euthanised and tissue samples were collected from the liver, glandular stomach and duodenum. The tissue samples were processed and scored for the presence of Comets which are indicative of DNA strand breakage in the assessed tissues.

The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity values in any of the tissues investigated when compared to the concurrent vehicle control group of the liver, glandular stomach or duodenum. The test item was considered to be unable to induce DNA strand breakage to the liver, glandular stomach or duodenum in vivo, under the conditions of the test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

BMI was assessed for the ability to cause gene mutation in Salmonella typhimurium and Escherichia coli. The study was conducted in accordance with GLP and to OECD guideline 471 (Huntingdon Life Sciences, 2012). In this main study 4 strains of Salmonella Typhimurium and one strain of Escherichia Coli were treated with test substance in the absence and in the presence of S9 mix. For the first test, concentrations tested were up to 5000 µg per plate. Results were negative for all strains at all concentrations therefore a second test was conducted. This employed the pre-incubation assay. Maximum concentrations were 500 µg/plate in the absence of S9 mix and 1500 µg/plate in the presence of S9 mix. Again results were negative for all strains at all concentrations. Therefore there was no evidence of mutagenic activity in this bacterial system under the test conditions employed. A further bacterial reverse mutation assay (Ames test) conducted at NOTOX (2008) further found no evidence of gene mutation in any of the Salmonella or E. Coli strains tested either in the presence or absence of metabolic activation, confirming the above conclusion.

A test was conducted (Safe Pharm Laboratories, 1988) in vitro in Chinese Hamster Ovary cells to assess the clastogenic potential of BMI in mammalian cells. The test was conducted using a 6-hour exposure period in the presence of metabolic activation (achieved by adding induced rat liver homogenate metabolising system) and a 24-hour exposure period in the absence of metabolic activation.

Significant dose-related increases in both toxicity and the frequency of aberrations were observed under both regimes of treatment with the test material. BMI was concluded to be clastogenic to CHO cells in vitro.

As the available in-vitro data indicated the possibility of a mutagenic effect, an in-vivo Comet assay was conducted (Envigo, 2018). Three groups of male rats received two doses of BMI (500, 1000 and 2000 mg/kg) approximately 24 hours apart. Following dosing the rats were humanely euthanised and tissue samples were collected from the liver, glandular stomach and duodenum. The tissue samples were processed and scored for the presence of Comets which are indicative of DNA strand breakage in the assessed tissues.

The test item did not induce any statistically significant increases in the percentage tail intensity or median percentage tail intensity values in any of the tissues investigated when compared to the concurrent vehicle control group of the liver, glandular stomach or duodenum. The test item was considered to be unable to induce DNA strand breakage to the liver, glandular stomach or duodenum in vivo, under the conditions of the test.

Overall it is concluded that although the in-vitro study in Chinese Hamster Ovaries indicated a clastogenic effect, the absence of any indication of a mutagenic effect in vivo indicates that BMI does not present any concern for a genotoxic effect in humans.

Justification for classification or non-classification

BMI was found not to have induced DNA strand breakage in an in vivo study assessing the effect in the glandular stomach, duodenum or liver of rats. On the basis of this lack of an effect BMI is considered not to meet the criteria for classification for Germ Cell Mutagenicity in accordance with Annex I section 3.5 of Regulation (EC) 1272/2008 (the CLP Regulation).