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Scheduling delegate's interim decisions and invitation for further comment: ACCS/ACMS, March and July 2017

Scheduling medicines and poisons

15 September 2017

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3.5 Bacillus amyloliquefaciens

Referred scheduling proposal

An application was submitted by the Australian Pesticides and Veterinary Medicines Authority (APVMA) to create a new entry for Bacillus amyloliquefaciens (B. amyloliquefaciens) in Schedule 5 of the Poisons Standard, with no exemption cut-off.

Scheduling application

This was a general application. The applicant's proposed amendments to the Poisons Standard are:

Schedule 5 – New Entry

BACILLUS AMYLOLIQUEFACIENS

The applicant's reasons for the request are:

  • B. amyloliquefaciens is a ubiquitous bacterium found in water, soil, air, decomposing plant material, on fresh produce and is widely used for the production of enzymes and specialty chemicals. B. amyloliquefaciens is described as being antagonistic towards fungal plant pathogens via nutrient competition, site exclusion and colonisation.
  • Based on the observed moderate eye irritation in rabbits, B. amyloliquefaciens strain QST 713meets the Scheduling Policy Framework (2015) criteria for scheduling it to Schedule 5 of the Poisons Standard with no cut-off or exemptions.
  • The acute inhalation toxicity LC50 =2150 mg/m3 4 h achieved for the formulated product was below the cut-off of 3000 mg/m3 4 h. However, no mortalities were observed at the achieved aerosol exposure and at necropsy, 2/10 rats had discoloured lungs and 8/10 rats were normal. Therefore, while the cut-off exposure of ≥ 3000 mg/m3 4 h was not achieved, the absence of mortalities and adverse clinical and macroscopic signs of toxicity at 2150 mg/m3/4 h suggest that this criterion was likely to have been achieved if the test was performed on the active constituent at 3000 mg/m3 4 h.
  • The formulated product was found to be positive for skin sensitisation and classified as 'May cause skin sensitisation by contact.' Therefore, in the absence of a study performed on the pure microbial preparation, it was also considered to be a skin sensitiser. The extent of skin sensitisation reported was considered to approximate the descriptor of 'slight' where scores were between 'no visible change' and 'discrete or patchy erythema.'
  • The potential for respiratory sensitisation has not been specifically tested. In a 4-week inhalation toxicity study, there was no clear evidence of irreversible systemic toxicity. In the statements from the applicant regarding worker exposure at two production plants, there was no indication of an increased incidence of respiratory illnesses/sensitisation reactions as a result of long-term exposure. However, limited mutagenicity, reproductive toxicity and developmental toxicity studies performed using substilin and/or surfactin C, the major endogenous protein metabolites produced by B. amyloliquefaciens did not indicate any toxicity findings of concern.
  • Repeat dose toxicity, carcinogenicity, genotoxicity, reproductive toxicity, developmental toxicity and neurotoxicity studies were not performed as it was a microbial preparation.

Current scheduling status and relevant scheduling history

B. amyloliquefaciensis not currently scheduled and has not been previously considered for scheduling. Therefore, a scheduling history is not available.

Australian regulatory information

B. amyloliquefaciensis not listed in the Therapeutic Goods (Permissible Ingredients) Determination No. 3 of 2017, and is not an excipient or active in any products on the ARTG.

International regulations

USA

The US EPA: B. amyloliquefaciens MBI 600 (antecedent Bacillus subtilis MBI 600) was registered in 1998; B. amyloliquefaciens strain D747 was registered in 2011; and B. amyloliquefaciens strain PTA-4838 was registered in 2017.

UK

B. amyloliquefaciens was approved in 2016 as a pesticide.

EU

B. amyloliquefaciens is currently in the pre-registration process.

Substance summary

B. amyloliquefaciens is a ubiquitous bacterium found in water, soil, air, decomposing plant material, on fresh produce and is widely used for the production of enzymes and specialty chemicals.

Table 3.5.1: Acute toxicity end-points for Bacillus amyloliquefaciens strain QST 713
Toxicity Species Bacillus amyloliquefaciens SPF (2015) Classification
Acute oral toxicity LD50(cfu/rat) Rat >1.13 x 108 Nil
Acute dermal toxicity LD50 (mg/kg bw) Rat >2000 Schedule 5
Acute inhalational toxicity LC50(cfu/rat) Rat >1.2 x 108 Nil
Skin irritation Rabbit Not irritant Nil
Eye irritation Rabbit Moderate Schedule 5
Skin sensitisation Not tested Not tested N/A

Acute toxicity

The acute toxicity of B. amyloliquefaciens QST 713 was low by oral, dermal and intra-tracheal administration (LD50 >1.13 x 108 cfu/rat, >2000 mg/kg bw and >1.2 x 108 cfu/rat respectively). B. amyloliquefaciens, QST 713 was a slight eye irritant but not a skin irritant. A skin sensitisation study was not performed on the microbial preparation.
Acute microbial pesticide evaluation also included an acute intravenous toxicity study for infectivity where the intravenous LD50 was >9.4 x 10 6cfu/rat, demonstrating low systemic infectivity/ pathogenicity potential (in rodents). No treatment-related mortalities occurred in any of the acute toxicity studies.

Skin irritation

There was no evidence of moderate or severe skin irritation caused by the test substance as assessed by the described methodology. Following the 4 h topical treatment period, very slight erythema was observed in 4/6 rabbits at 30-60 min, and 3/6 rabbits at 24 h after removal of the semi-occlusive dressing.

The test substance is considered a non-skin irritant due to the absence of irritation at 72 h.

While the study was not considered to be equivalent to a test guideline compliant study and sufficient to support formal classification of skin irritancy potential, the APVMA considers that the test substance would be unlikely to be a skin irritant.

Eye irritation

There was no evidence of persistent moderate or severe eye irritation caused by the test substance.

Following treatment, conjunctival redness grade 2 was present in 6/6 rabbits at 1 h and 1/6 rabbits at 24 h post dosing and persisted as grade 1 in 3/6 rabbits at 72 h. Conjunctival chemosis occurred 6/6 rabbits ranging from grade 1 to grade 3 at 1 h, and decreased to grade 1 in 1/6 rabbits at 24 h.

The test substance is considered to be a slight eye irritant due to the presence of slight irritation without corneal opacity.

While the study was not considered to be equivalent to a test guideline compliant study, the APVMA considers that the test substance would be likely to be a slight eye irritant.

Repeat-dose toxicity

Repeat dose toxicity studies were not conducted based on the absence of findings in the acute toxicology and infectivity/pathogenicity studies. This is reasonable and appropriate for a non-pathogenic biological active ingredient of this type. A 4-week inhalation study was performed in rats in which no toxicologically significant adverse effects were observed at the single dose level tested.

Supportive evidence for minimal short-term toxicity was provided by a 28-day oral toxicity of surfactin C rats (Hwang et al., 2009). No clinical signs of toxicity, alterations in body weight, body weight gain and food consumption, alterations on the haematological parameters, differences in organ weights or histopathological findings were noted at 500 mg/kg bw/d. Decreased bodyweight, increased alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, increased liver weights and hydrophic necrosis of hepatocytes were observed at higher doses.

Genotoxicity

No evidence of genotoxicity has been shown to be produced by B. amyloliquefaciens.

Carcinogenicity

No information was provided.

Reproduction and developmental toxicity

No evidence of reproduction or developmental toxicity has been shown to be produced by B. amyloliquefaciens.

Clinical observations

EFSA conducted a review update of the safety concerns for the Qualified Presumption of Safety (QPS) status of biological agents that are added intentionally to food and animal feeds including Bacillus species (EFSA, 2013). The observations reported and summarised in the QPS document were considered clinically relevant to the current assessment and addressed reports of claims where B. amyloliquefaciens was a causative agent in clinical cases. The relevant QPS review text is presented below (with minor edits). Collectively, there was no substantive new evidence that confirmed that B. amyloliquefaciens was the causative agent in the clinical reports and also confirmed the existing EFSA position of non-toxigenic/non-pathogenic potential of B. amyloliquefaciens towards humans.

In total, 230 articles found by relevant search terms were screened. A bacteraemia related to a pacemaker wire infection was caused by B. licheniformis (Idelevich et al., 2012). B. amyloliquefaciens and B. licheniformis were identified as the cause of a bacteraemia in a patient with an oesophageal perforation (La Jeon et al., 2012). Kim et al., (2012) reported a case of bacteraemia caused by B. licheniformis following vertebrotherapy in a patient with lung cancer. Safety concerns for food producing animals were also considered in the search because 'the body of knowledge about the organisms for which QPS is sought must be sufficient to provide adequate assurance that any potential to produce adverse effects in humans, livestock or the wider environment is understood and predictable' (EFSA, 2007). A Bacillus sp. was isolated from abscesses in several sheep and goats, but authors could not identify the isolates to the species level by phenotypic tests and sequence of 16s rRNA gene (Mariappan et al., 2012). Gangrenous mastitis in several goats was caused by Bacillus spp., one of the isolates was identified B. cereus, but other isolates were not identified at the species level (Mavangira et al., 2013). B. amyloliquefaciens was isolated, together with staphylococcus, from milk of goats with subclinical mastitis (Razi et al., 2012), but without evidence that B. amyloliquefaciens was the cause of the mastitis.

These infections in humans were linked to specific predisposing factors and did not suggest a risk for the consumer via exposure through the food and feed chain. The abscesses reported in sheep were not sufficiently characterised to determine whether Bacillus species from the QPS list were involved. In respect to the report of mastitis in goats, the co-isolation of S. aureus, a well-known agent of mastitis, raised doubt regarding the role of B. amyloliquefaciens in the infection.

Public exposure

A review article on foodborne illness(es) caused by Bacillus species, including some QPS Bacillus species was published in 2012 (Logan, 2012). The outcomes of the review were in line with the previous QPS assessment (EFSA, 2008) concerning the rare implication of QPS Bacillus species in foodborne illnesses, and the likely implication of peptidolipids with toxic activities produced by the responsible strains. Two articles described some biological activities of peptidolipids with biosurfactants produced by B. amyloliquefaciens. A biosurfactant produced by a strain of B. amyloliquefaciens caused epithelium cell vacuolisation and microvilli damage in the mid-gut of an insect larvae (LC50 approx. 200 ng/mg according to Ghribi et al., 2012) and a B. amyloliquefaciens strain isolated from a Korean fermented soybean paste produced up to 48 mg surfactin per kg in the fermented food, and the surfactin inhibited growth of human breast cancer cells (IC50 10 μg/mL, Lee et al., 2012).

The applicant identified a range of clinically relevant publications in which B. amyloliquefaciens was associated with a range of disease manifestations (Ochoa, 2015, Aoki et al., 2015; Baur & Bakehe, 2014; Hong et al., 2008; Inomata et al., 2007; Long et al., 2014; Pavic et al., 2005; Stickel et al., 2008). Collectively, these publications identified B. amyloliquefaciens as being present along with other possible co-causative agents. The case subjects (human and domestic/farm animals) often had pre-existing conditions or a reasonable likelihood of compromised immune system function. B. amyloliquefaciens, QST 713 was not identified as being associated with a clinical condition in the citations and no citation suggested an association between B. amyloliquefaciens with genotoxic, carcinogenic or reproductive toxicity potential. Broadly, the citations were consistent with the EFSA QPS review summary discussed above.

Several citations have investigated allergenic or hypersensitivity reactions in individuals who have been repeatedly exposed B. amyloliquefaciens and such responses have been attributed to exposure to the extracellular enzyme subtilisin (EPA 1997) and may be consistent with the slight skin sensitisation response observed for the formulated product. However, the applicant provided statements for the absence of an increased incidence in allergy/respiratory related concerns in B. amyloliquefaciens QST 713 manufacturing facilities (Miille, 2015; Navarro, 2015). In addition, ELISA testing of the QST 713 strain for subtilisin was reported to be negative (Manker, 2002, Chemistry and Manufacturing submission, APVMA data No. 116454) indicating that the sensitisation response in animals is towards another formulation component.

Antibiotic susceptibility testing against common human antibiotics provided supportive evidence for the absence of resistance genes in B. amyloliquefaciens QST 713 with the exception of bacitracin (Lehman, 2001, 2002).

Pre-meeting submissions

No public submissions were received.

Summary of ACCS advice to the delegate

The committee recommended that a new Appendix B entry for Bacillus amyloliquefaciens strain QST 713 be created as follows:

Appendix B – New Entry

BACILLUS AMYLOLIQUEFACIENS STRAIN QST 713.

Reason for Entry – a, low toxicity

Area of Use – 1.3, fungicide

The committee recommended that a cross-reference in the Index for Bacillus subtilis QST 713 be created as follows:

Index – New Entry

BACILLUS AMYLOLIQUEFACIENS STRAIN QST 713
cross reference: BACILLUS SUBTILIS STRAIN QST 713

The committee also recommended an implementation date of 1 February 2018.

Members agreed that the relevant matters under Section 52E(1) of the Therapeutic Goods Act 1989 included: (a) risks and benefits of the use of a substance; and (c) the toxicity of a substance.

The reasons for the advice were:

  • The risks posed by Bacillus amyloliquefaciens strain QST 713 are very low. There may be a small risk to groups susceptible to infection.
  • There are a range of benefits for preventing fungal disease in certain food crops.
  • Bacillus amyloliquefaciens is a naturally occurring microorganism with very low infectivity, low pathogenicity and a low risk of causing skin irritancy. The risk is mitigated by personal protective equipment worn on mixing/loading and application in accord with APVMA labels.
  • Worker data shows that Bacillus amyloliquefaciens strain QST 713 is unlikely to be a respiratory sensitiser.

Delegate's considerations

The delegate considered the following regarding this proposal:

  • Scheduling proposal
  • ACCS advice
  • Section 52E of the Therapeutic Goods Act 1989
  • Scheduling Policy Framework (SPF 2015)
  • Other relevant information

Delegate's interim decision

The delegate's interim decision is to create a new Appendix B entry for Bacillus amyloliquefaciens QST 713, with an index cross reference for Bacillus subtilis Strain QST 713. The proposed Schedule entry is as follows:

Appendix B – New Entry

BACILLUS AMYLOLIQUEFACIENS STRAIN QST 713.

Reason for Entry – a, low toxicity

Area of Use – 1.3, fungicide

Index – New Entry

BACILLUS AMYLOLIQUEFACIENS STRAIN QST 713
cross reference: BACILLUS SUBTILIS STRAIN QST 713

The proposed implementation date is 1 February 2018, as this is the earliest possible implementation date.

The matters under subsection 52E(1) of the Therapeutic Goods Act 1989 considered relevant by the delegate included: (a) the risks and benefits of the use of a substance; and (c) the toxicity of a substance.

The reasons for the interim decision are:

  • The risks posed by Bacillus amyloliquefaciens strain QST 713 are very low. There may be a small risk to groups susceptible to infection.
  • There are a range of benefits for preventing fungal disease in certain food crops.
  • Bacillus amyloliquefaciens is a naturally occurring microorganism with very low infectivity, low pathogenicity and a low risk of causing skin irritancy. The risk is mitigated by personal protective equipment worn on mixing/loading and application in accord with APVMA labels.
  • Worker data shows that Bacillus amyloliquefaciens strain QST 713 is unlikely to be a respiratory sensitiser.

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