Registration Dossier

Administrative data

Description of key information

The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 (adopted 1998), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, equivalent to 10 mL of undiluted test material/kg bw/day or 1248.3 mg enzyme concentrate dry matter/kg bw/day.

The repeated dose inhalation and dermal toxicity studies were waived.

- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme that toxicologically relevant is unrealistic, due to the formulation of enzymes and the stringent work practices, enforced because of the risk of sensitisation by inhalation.

Key value for chemical safety assessment

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:
October 26, 2006 – August 7, 2007
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:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Rat, SPF Sprague Dawley of the Ntac:SD strain from Taconic Europe A/S, Ejby, Denmark
- Age (at Delivery): 5 weeks
- Body Weight Range (at Acclimatization): within +/- 20 rams for each sex.
- Housing: Animal room no. 118 in transparent polycarbonate cages (floor area: 1500 cm2) with two or three in each cage, males and females separated.
- Diet (e.g. ad libitum): A complete pelleted rodent diet “Altromin 1314 Fortified” (for growing animals) was available ad libitum until Day 53 of the dosing period. On Day 54 and throughout the study, animals were offered ad libitum “Altromin 1324 Fortified” (for adult animals).
- Water: Acidified domestic quality drinking ad libitum
- Acclimation period: At least 5 days in order to reject animals in poor condition or at the extremes of the weight range.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%):55 ± 15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hours light

IN-LIFE DATES: Animals arrived on 22 November 2006. Treatment started on 29 November 2006. In-life phase ended on 01 March 2007.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Lipase was administered daily by oral gavage in tap water (vehicle), dose volume was 10 mL/kg.

Dose levels were 0 (vehicle), 124.8 (low), 411.94 (medium) and 1248.3 (high) mg/kg body weight/day based on enzyme concentrate dry matter for a period of 13 weeks.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The measured concentration of the dosing solutions expressed in enzymes activity units per g was found close to the intended content of the test material formulations for the high, medium and low dose groups in week 1, 6 and 13.


Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
given in mg enzyme concentrate dry matter
Dose / conc.:
124.8 mg/kg bw/day (nominal)
Remarks:
given in mg enzyme concentrate dry matter
Dose / conc.:
411.9 mg/kg bw/day (nominal)
Remarks:
given in mg enzyme concentrate dry matter
Dose / conc.:
1 248.8 mg/kg bw/day (nominal)
Remarks:
given in mg enzyme concentrate dry matter
No. of animals per sex per dose:
10 female and 10 male rats
Control animals:
yes, concurrent vehicle
Details on study design:
Doses were selected based on previous experience with lipase in 13 wk oral gavage toxicity studies in rats.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
At least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical signs were recorded daily and detailed clinical observations were performed outside the home cage once a week.

BODY WEIGHT: Yes
Animals were weighed on the day of arrival, on the first day of treatment (Day 1) and weekly thereafter.

FOOD CONSUMPTION: Yes
Starting Day 1, the consumption of food was recorded weekly for each cage

WATER CONSUMPTION: Yes
The consumption of water was recorded twice weekly for each cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
Before treatment started. Before termination of treatment, animals in group 1 and 4 were to be re-examined; however, as a deviation to the study plan, this was not done.

CLINICAL CHEMISTRY: Yes

HEMATOLOGY: Yes

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Weekly observations included observations for changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling) or bizarre behaviour (e.g., self-mutilation, walking backwards). Moreover, on one occasion during the last two weeks of the study, all animals were examined in the Open field and stimuli-induced test and were observed with respect to reactivity to different types of stimuli (e.g. auditory, visual, tactile), grip strength and motor activity (open field test).
Sacrifice and pathology:
At the end of the treatment period (week 13), blood samples were withdrawn for haematology and plasma biochemistry analyses. All animals were then sacrificed in CO2/O2. At necropsy a bone marrow smear was taken from the femur of all animals. Gross pathology was performed and histopathology was performed in the control group (Group 1) and in the high dose group (Group 4).
Other examinations:
Weight of individual organs: yes
Statistics:
Data were processed to give group mean values and standard deviations where appropriate.
Thereafter each continuous variable was tested for homogeneity of variance with Levene’s test. If the variance was homogeneous, analysis of variance was carried out for the variable. If any significant differences were detected, possible inter-group differences were assessed with Dunnett’s test (comparing treated groups with a control group). If the variance was heterogeneous, each variable was tested for normality by the Shapiro-Wilk method. In case of normal distribution, possible inter-group differences were identified with Student's t-test.
Otherwise the possible inter-group differences were assessed by Kruskal-Wallis's test. If any significant inter-group differences were detected, the subsequent identification of the groups was carried out with Wilcoxon Rank-Sum test. For all tests, the level of significance was defined as p<0.05. The statistical analyses were made with SAS® procedures (version 8.2) described in "SAS/STAT® User's Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.
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:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The haematological findings were considered incidental findings.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
The clinical chemistry findings were considered incidental findings
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The neurobehavioural findings were considered incidental findings
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 248.3 mg/kg bw/day (nominal)
Based on:
other: Enzyme concentrate dry matter
Sex:
male/female
Basis for effect level:
other: There were no adverse effects observed at the highest does tested.
Key result
Critical effects observed:
no
Conclusions:
Oral administration (gavage) of Lipase to Sprague-Dawley rats at doses up to 1248.3 mg enzyme concentrated dry matter/kg/day for a period of 13 weeks was generally well tolerated.

No test item-related deaths occurred and no changes in daily or weekly observations as well as functional observational battery (performed during week 13) including grip strength and locomotor activity were observed. Furthermore, no test item-related effects on body weights or food consumption, no ophthalmoscopic findings, no changes in clinical laboratory investigations of toxicological relevance as well as no macroscopic and microscopic findings were noted.

Based on the results of this study, the no observed adverse effect level (NOAEL) for the lipase was set at 1248.3 mg enzyme concentrate dry matter/kg/day.
Executive summary:

The repeated dose oral toxicity study was a 13-week subchronic toxicity study conducted in rats according to OECD guideline No. 408 (revised in 1998) and in compliance with GLP.

The test material used in this study, the lipase (batch no. PPW 26090) was dissolved in tap water. The test material was given to 10 animals/sex/group in a constant volume of 10 mL/kg bw by gavage to achieve the desired concentrations of 124.8, 411.9 and 1248.3 mg enzyme concentrated dry matter/kg bw/day for 90 consecutive days. Tap water given at the same volume served as vehicle control. The test material in solution was tested for stability, and concentration in dosing solutions was verified by analysis. Clinical observations were made daily whereas feed and body weight were recorded weekly. Clinical chemistry, hematology, open field and stimuli-induced tests, necropsy, organ weights, and macroscopic- and histopathological examinations were conducted at study termination as required by the guideline. One deviation to the guideline was that ophthalmoscopy by mistake was not performed before termination. However, the histopathological examination revealed no pathological findings in the eyes of any of the animal.

 

There were no treatment related effects found in any of the parameters investigated.

In summary, oral administration of lipase to rats of both sexes at dose levels up to 1248.3 mg enzyme concentrate dry matter/kg bw/day for 13 consecutive weeks did not result in any systemic, behavioural or pathological changes. 

The No Observed Adverse Effect Level (NOAEL) was therefore established at the highest dose tested, 1248.3 mg enzyme concentrate dry matter/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 248.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Data waiving:
exposure considerations
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 (adopted 1998), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, equivalent to 10 mL of undiluted test material/kg bw/day or 1248.3 mg enzyme concentrate dry matter/kg bw/day.

The repeated dose inhalation and dermal toxicity studies were waived.

- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme that toxicologically relevant is unrealistic, due to the formulation of enzymes and the stringent work practices, enforced because of the risk of sensitisation by inhalation.

Based on repeated dose oral and weight of evidence, lipase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.

Justification for classification or non-classification

Based on repeated dose oral study and weight of evidence, lipase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.