1,4-α-glucan branching enzyme

Endpoint summary

Description of key information

The highest concentration (undiluted concentrate) of 1,4-alpha-glucan branching enzyme, batch PPY 27209 did not cause any adverse changes in any of the investigated parameters in the sub-chronic toxicity study in rats. Therefore, the no-observed-adverse-effect level (NOAEL) for 1,4-alpha-glucan branching enzyme, batch PPY 27209 was considered to be 1128 mg Dry matter/kg bw/day for both sexes.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 July 2007 to 13 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This assessment of sub-chronic systemic toxicity (according to OECD TG 408) has been performed due to data requirements from the European Food Safety Authority (EFSA), as this enzyme also comply with the regulatory system of FIAP [REGULATION (EC) No 1331/2008 and EFSA CEF guidance from 2009/2013]. Moreover; it has been generated in accordance with Directive 2010/63/EU.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 21st September 1998.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: approximately 6 week
- Weight at study initiation: Weighed 135.2 g (mean for females) and 179.6 g (mean for males)
- Fasting period before study: None
- Housing: In Macrolon cages (480 x 375 x 210 mm) with sterilized wood shavings (Lignocel, Type 3/4) as bedding material and strips of paper (Enviro-dri) as environmental enrichment.
- Diet: Rat & Mouse No. 3 Breeding Diet, RM3; pelleted
- Water: Ad libitum. Water, taken from the public supply.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity : 40-70% RH
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

and purified water for the other control

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle were prepared weekly and stored in closed vials (one vial per group per day) in a freezer at < -18ºC.
VEHICLE
- The vehicle for dosing the controls was stored as described above.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations prepared for administration on one occasion in Week 1 and 13 were taken for content check analysis. The results of these analyses indicated values close and acceptable to the intended enzyme activity.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

113 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Enzyme concentrate dry matter.

Dose / conc.:

372 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Enzyme concentrate dry matter.

Dose / conc.:

1 128 mg/kg bw/day (nominal)

Remarks:

Concentration is in mg Enzyme concentrate dry matter.

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The highest dose (100%) is the maximum practical dose and represents administration of the enzyme, as received, at a volume dosage of 10 mL/kg body weight.

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual observations of all animals were also recorded before and after dosing on each day of treatment as indicated below. The timing of these observations were performed to establish and confirm any pattern of signs.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded during the acclimatisation period, on the day that treatment commenced, at weekly intervals throughout the treatment period and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each cage, that remaining and an estimate of the amount spilled was recorded for each week throughout the treatment period. From these records the mean weekly consumption per animal was calculated for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Were made prior to the start of treatment in all rats, and in the last week of the treatment period in all rats of the control group and the high-dose group.
- Dose groups that were examined: Observations were bilateral unless otherwise indicated.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
Differential leucocyte counts were determined automatically by counting the numbers of lymphocytes, neutrophils, monocytes, eosinophils, basophils and large unstained cells in the instrument sample.

CLINICAL CHEMISTRY: Yes
The concentration of each protein fraction was determined by reference to the percentage value and to the total protein concentration. Albumin to globulin (A/G) ratios were calculated from the percentage values before conversion to absolute concentrations.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
ORGAN WEIGHTS: Yes

Statistics:

The main tests used were analysis of covariance (ANOVA), Shapiro-Wilks test, Dunnett's multiple comparison test, Kruskal-Wallis non-parametric analysisi, Pearson chisquare analysis, and Fisher's Exact Probability test. Significant differences between the groups compared were expressed at the 5% (p<0.05) level.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Only a significantly lower mean corpuscular haemoglobin concentration (MCHC) in high-dose males were observed. This change was not seen in females and was not estimated to be test item related.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

The following statistically significant differences in organ weights were observed compared to controls: 1) Absolute brain weight was significantly lower in males of the high-dose group, 2) Relative liver weight was significantly higher in males of the high-dose group and 3) Absolute and relative weight of the epididymides was significantly lower in the mid-dose group. None of these observations were considered test item related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significantly increased incidence of renal basophilic tubules was observed in mid-dose males. Since basophilic tubules are part of common background pathology in (particularly male) rats with rather large variations in incidence, and a comparable finding was not observed in high-dose males or treated females, this finding was not considered to be related to the test-substance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 128 mg/kg bw/day (nominal)

Based on:

other: Enzyme concentrate dry matter

Sex:

male/female

Basis for effect level:

other: No adverse effects were seen so NOAEL was the highest dose administered.

Critical effects observed:

no

Conclusions:

Undiluted concentrate of 1,4-alpha-glucan branching enzyme, batch PPY 27209 did not cause any adverse changes in any of the investigated parameters in the sub-chronic toxicity study in rats. Therefore, the no-observed-adverse-effect level (NOAEL) for 1,4-alpha-glucan branching enzyme, batch PPY 27209 was considered to be 1128 mg Enzyme concentrate dry matter/kg bw/day for both sexes.

Executive summary:

The objective of this study was to provide data on the possible sub-chronic toxicity of 1,4-alpha-glucan branching enzyme, batch PPY 27209 in rats of both sexes upon daily oral administration by gastric gavage for 13 consecutive weeks. Rats were exposed to 113, 372, 1128 mg Enzyme concentrate dry matter/kg bw/day.

Clinical observations, neurobehavioural testing, growth, food and water consumption, ophthalmoscopy, haematological and clinical chemistry parameters, gross examination at necropsy, organ weights and microscopic examination of various organs and tissues were used as criteria for disclosing possible harmful effects.

No indication of toxicity of the test substance was observed after oral adminitration in rats, since none of the parameters investigated showed any treatment-related or toxicologically relevant changes.

In conclusion, undiluted concentrate of 1,4-alpha-glucan branching enzyme, batch PPY 27209 did not cause any adverse changes in any of the investigated parameters in the sub-chronic toxicity study in rats. Therefore, the no-observed-adverse-effect level (NOAEL) for 1,4-alpha-glucan branching enzyme, batch PPY 27209 was considered to be 1128 mg Enzyme concentrate dry matter/kg bw/day for both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 128 mg/kg bw/day

Study duration:

subchronic

Species:

rat