Pullulanase

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 28 - August 3, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ICH S4A - Single dose toxicity

Version / remarks:

1991

Deviations:

no

Principles of method if other than guideline:

A limit acute oral toxicity test was performed. A preliminary group of one male and one female rat received a single dose of the undiluted test material in a volume of 20 mL/kg body weight. These animals were observed for 7 days and since the dosing was well tolerated, additionally five males and females were dosed at 20 mL/kg body weight followed by a 14 days observation period, where clinical conditions and bodyweight were followed. At the end, the animals were killed and macroscopic examination was performed.

GLP compliance:

yes

Test type:

other: ICH S4A - Single dose toxicity, 1991

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Pullulanase

EC Number:

232-983-9

EC Name:

Pullulanase

Cas Number:

9075-68-7

Molecular formula:

not available

IUPAC Name:

Pullulanase

Constituent 2

Reference substance name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available

IUPAC Name:

Protein as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 3

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

Reference substance name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Test material form:

liquid

Details on test material:

- Substance type: UVCB
- Physical state: Liquid
- Lot/batch No.: PPY 25645
- Expiration date of the lot/batch: 09 March 2016
- Stability under test conditions: The test material and dilutions are stable for at least 24 hours at 5 degrees of C and at room temperature
- Storage condition of test material: Approximately minus 20 degrees of Celcius. Thawed and refrozen on no more than one occasion.

Test animals

Species:

rat

Strain:

other: Crl:CD® (SD)IGS BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River (UK) Ltd
- Age: 35 to 41 days at the day of treatment
- Fasting period before dosing: overnight fasting prior to dosing
- Housing: Barriered rodent facility with control of temperature and humidity. Five of the same sex in cages made of a polycarbonate body and floor with a stainless steel mesh lid. Lignocel type 3/4 wood shavings were provided as bedding. Aspen chew block for environmental enrichment.
- Weight at the end of the acclimatisation period: Males 149 - 168 g, females 116 -130 g.
- Diet: Pelleted standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) ad libitum
- Water: Water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Undiluted test material

Doses:

Dose volume was 20 mL/kg bodyweight (equivalent to 2.012 g TOS/kg bodyweight, 31230 PUN-RPA/kg)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: soon after dosing, at least twice during the first hour after dosing (approximately 30 minutes apart) and subsequently at approximately one-hour intervals for the remainder of day 1. On subsequent days animals were inspected at least twice daily. Weighing was done immediately before dosing at day 1, and on day 8 and day 15.
- Necropsy of survivors performed: yes

Statistics:

No

Results and discussion

Effect levels

Effect levels 1

Key result

Sex:

male/female

Dose descriptor:

other: Fixed dose method - no effects were seen

Effect level:

> 20 mL/kg bw

Based on:

test mat.

Effect levels 2

Key result

Sex:

male/female

Dose descriptor:

other: Fixed dose method - no effects were seen

Effect level:

> 2 012 mg/kg bw

Based on:

other: Total Organic Solids (TOS)

Mortality:

No animals died during the study.

Clinical signs:

No animals died and no sign of toxicity or ill health was seen at the routine physical examination.

Body weight:

No effect was observed on the body weights.

Gross pathology:

Effects on organs:
Macroscopic examination of animals killed on Day 15 of the observation period did not reveal any treatment-related findings.

Other findings:

Punctate clear cysts were found on the left kidney of female No. 11. This is a common finding in laboratory rodents and was considered to be unrelated to a single administration of Pullulanase, PPY 25645.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of toxicity were observed among the rats treated with a single oral dose of 2012 mg total organic solids/kg, which was the highest possible dose at dose volume 20 mL/kg, using the undiluted test item.

Executive summary:

The study was conducted as a limit test in accordance with the International Conference on Harmonisation (ICH) S4A – Single dose toxicity guidance, which is principally similar to the OECD Guideline No 420, "Acute Oral Toxicity - Fixed dose Method".

The test item was supplied as a brown liquid. The dose volume administered was 20 mL of the undiluted test material per kg body weight, corresponding to a dosage of 2.012 g Total Organic Solids (TOS)/kg bw. A group of five male and five female rats received the test material by a single oral administration (gavage). The animals were subjected to clinical observations daily for a fourteen day observation period and at termination of the study, gross necropsy of all animals was carried out.

 

No clinical effects were observed and the overall body weight gain during the study was considered to be normal. The post-mortem inspection revealed no abnormalities.

In conclusion, no signs of toxicity were observed among the rats treated with a single oral dose of 20 mL of the undiluted test material per kg body weight (= 2.012 g TOS/kg bw).