  SEQ CHAPTER \h \r 1 Primary Reviewer:	1288	Date: April 12, 2016

	Jonas Ptasinski

Senior Evaluation Officer

Microbial and Biochemical Evaluation Section (MBES), Health Effects
Division I (HED I)

Health Evaluation Directorate (HED)

	  SEQ CHAPTER \h \r 1 Approved by:	203	Date: April 14, 2016

	Brian Belliveau

Head, MBES, HED I, HED

	_____________

  SEQ CHAPTER \h \r 1 REQUIREMENT:	PMRA Data Code:		M4.2.3–Acute
Pulmonary Infectivity and Toxicity 

			U.S. EPA OPPTS Guideline: 	885.3150

			OECD Data Code:		IIM 5.3.3

  SEQ CHAPTER \h \r 1 TEST MATERIAL (PURITY):	Bacillus mycoides isolate
J TGAI (9.3 × 1010 colony forming units [CFU]/g [nominal]; 1.6 × 1011
CFU/g [enumerated])

SYNONYMS:	  SEQ CHAPTER \h \r 1 none

CITATION:	  SEQ CHAPTER \h \r 1 Doig, A. 2016. Bacillus mycoides Isolate
J TGAI Acute Inhaltation Toxicity/Pathogenicity in Rats, Additional
Confirmatory Testing. Stillmeadow Inc. Laboratory report number
19791-16, February, 29, 2016. Unpublished. PMRA 2619368.

SPONSOR:	  SEQ CHAPTER \h \r 1 Certis USA LLC

COMPLIANCE:	Signed and dated GLP, Quality Assurance, and Data
Confidentiality statements were not provided. The study was conducted in
compliance with GLP guidelines. This DER does not contain CBI.

EXECUTIVE SUMMARY:	  SEQ CHAPTER \h \r 1 In an acute pulmonary toxicity
study, groups of 9 week old Sprague-Dawley rats (3/sex) were exposed by
the intratracheal route to Bacillus mycoides isolate J TGAI (1.6 × 1011
CFU/g) in 0.1 mL of phosphate buffered saline at a dose of 108
CFU/animal.  Animals were then observed for up to 48 hours. 

The 48-hour acute pulmonary LD50 for rats is > 108 CFU/animal

  SEQ CHAPTER \h \r 1 Based on these results, is of LOW Toxicity in the
rat. 

  SEQ CHAPTER \h \r 1 There were no treatment related clinical signs or
changes in body weight.  

CLASSIFICATION:	ACCEPTABLE 

I.	MATERIALS AND METHODS

A.	GUIDELINE FOLLOWED:	U.S. EPA OCSPP 885.3150: Acute Pulmonary 						
Toxicity/Pathogenicity  SEQ CHAPTER \h \r 1 

B.	MATERIALS:

1.	Test Material:	Bacillus mycoides isolate J TGAI

	Description:	brown powder

	Lot/batch #:	CX10244-3

	Purity:		9.3 × 1010 CFU/g (nominal); 1.6 × 1011 CFU/g (enumerated)

	Storage conditions:	room temperature

2.	Test Animals:

	Species:	rat

	Strain:		albino Sprague-Dawley

♂ 290 – 295 g; ♀: 187 – 209 g

	Source:	Texas Animal Specialties

C.	STUDY DESIGN AND METHODS:

1.	Experimental Methods and Conditions:

	In life dates:			Start:	February 15, 2016	End:	February 17, 2016

	Preliminary challenge assay:	  SEQ CHAPTER \h \r 1 none

	Acclimation:	5 day quarantine

	Housing:	polycarbonate box with bedding

	Diet:		PMI Feeds Inc. Formulab #5008 ad libitum

	Water:		municipal water ad libitum

	  SEQ CHAPTER \h \r 1 Animal assignment and treatment:	Animals were
assigned to the test groups noted in Table 1.   SEQ CHAPTER \h \r 1 The
dosing solution was administered via an appropriate delivery system in a
single dose into the trachea of each animal (3 males and 3 females).
Using a constant dose volume of 0.1 mL, each rat received approximately
108 CFU/rat.

TABLE 1.	 Doses, mortality/animals treated.

Test Group	Test Substance	Dose Level	Males	Females	Combined

1	Bacillus mycoides isolate J TGAI	♀: 0.1 mL (108 CFU)/rat

♂: 0.1 mL (108 CFU)/rat	0/3	0/3	0/6



	Sample preparation:	Prior to administration, the test substance was
analyzed to quantify the number of viable test microbes (CFU/g). An
aliquot (1.0 g) of test substance was dissolved in 9 mL of phosphate
buffered saline (PBS), serially diluted, plated on nutrient agar and
incubated at 28°C for approximately 21 hours until colonies were of
sufficient size to count. The nutrient agar was prepared by dissolving
approximately 19.5 g of nutrient agar into 850 mL of deionized water.
The PBS was prepared by dissolving 2 PBS tablets into 1000 mL of
deionized water. The dosing solution was also analyzed for CFU/mL using
the same plate count method. The CFU/g or mL was determined by the
following formula: (Mean Plate × Dilution Factor) / Volume Plated. 

	The test substance dosing solution was prepared by adding 1.0 g of test
substance to 9 mL PBS to make a 10-1 dilution. From the 10-1 dilution, 3
mL of solution were added to 27 mL of PBS for a 10-2 dilution, which
created a solution containing approximately 109 CFU/mL. 

	Controls:	none  SEQ CHAPTER \h \r 1 

	Environmental conditions:	Temperature	21 – 23°C 

					Humidity	33 – 77%

					Air changes	at least 10/h

					Photoperiod	12h dark/  12h light

	Solvent/vehicle: 	PBS

	Microbial enumeration:	  SEQ CHAPTER \h \r 1 n/a

	Sensitivity of detection:	  SEQ CHAPTER \h \r 1 n/a

	Duration of study:	48 hours

	Other methods or conditions, if any:	none

2.	Observations:

	  SEQ CHAPTER \h \r 1 Clinical observations and body weights:
Observations for mortality and clinical signs of toxicity effects were
made three times following dosing and daily thereafter for the duration
of the study. The nature, onset, severity and duration of all gross or
visible pharmacologic or toxicologic signs were recorded. Cage-side
observations included, but were not limited to: the skin and fur; eyes
and mucous membranes; respiratory, circulatory, autonomic and central
nervous systems; somatomotor activity and behavior patterns. Particular
attention was given to tremors, convulsions, diarrhea, lethargy,
salivation, sleep and coma. Body weights were recorded on the day of
dosing (Day 0) and at the final sacrifice, approximately 48 hours after
dosing.

	

	Feed consumption:	  SEQ CHAPTER \h \r 1 Feed consumption was not
measured.

	Necropsy and organ weights: 	Not performed

	Microbial enumeration: n/a

	Were raw data included?	Test material enumeration, body weight and
clinical observation data were included.

	Other observations, if any:	none

II.	RESULTS

A.	MORTALITY is given in Table 1. There were no mortalities.

	The pulmonary LD50 for	 males and female rats is > 108 CFU/animal.

						

B.	CLINICAL OBSERVATIONS:	  SEQ CHAPTER \h \r 1 There were no clinical
sings noted in any animal.

C.	BODY WEIGHT:	  SEQ CHAPTER \h \r 1 All animals gained weight in the
48-hour study duration, except for one male which remained at the same
weight as on Day 0.

D.	FEED CONSUMPTION:	  SEQ CHAPTER \h \r 1 n/a

E.	NECROPSY:	  SEQ CHAPTER \h \r 1 n/a

F.	ORGAN WEIGHTS:	  SEQ CHAPTER \h \r 1  n/a

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H.	REPORTED STATISTICS:	n/a

III.	CONCLUSION

	The study author concluded that “The test substance, Bacillus
mycoides isolate J TGAI, was determined to be non-toxic to rats when
administered by tracheal injection in a single dose of 1.8 × 109 CFU/mL
or 1.8 x 108 CFU/rat.

A.	REVIEWER’S CONCLUSIONS:	  SEQ CHAPTER \h \r 1 The reviewer
concludes that the 48-hour tracheal instillation LD50 for Bacillus
mycoides isolate J TGAI is > 108 CFU/rat.

B.	DEFICIENCIES:   SEQ CHAPTER \h \r 1 	none

IV.	REFERENCES	  SEQ CHAPTER \h \r 1 none

Sub. No. 2014-1243 / Company code CIT

