Catalase

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 8, 2014 to March 30, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed according to OECD Guideline 408 (1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is also used for production of food in EU.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of catalase (EC no. 232-577-1, CAS no. 9001-05-2, EC name Catalase, Enzyme class no. 1.11.1.6)

Molecular formula:

Not applicable, see remarks

IUPAC Name:

Active enzyme protein of catalase (EC no. 232-577-1, CAS no. 9001-05-2, EC name Catalase, Enzyme class no. 1.11.1.6)

Constituent 2

Reference substance name:

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

Molecular formula:

Not available. See remarks.

IUPAC Name:

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

Constituent 3

Reference substance name:

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

Molecular formula:

Not available. See remarks.

IUPAC Name:

Lipids 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. See remarks.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process.

Constituent 5

Reference substance name:

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

Molecular formula:

Not available. See remarks.

IUPAC Name:

Inorganic salts 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: light brown liquid
- Stability under test conditions: The neat test substance was demonstrated to be stable under the conditions of the study.

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat was selected for this study because it is the recommended species specified in the guidelines. The Crl:CD(SD) rat was selected based on consistently acceptable health status and on extensive experience with this strain at the performing laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories International, Inc., Raleigh, North Carolina, U.S.A.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 49 days old
- Weight at study initiation: 280±10 grams
- Housing: Animals were housed in pairs in solid-bottom caging with bedding and appropriate species-specific enrichment. Each cage rack contained only animals of one sex.
- Diet (e.g. ad libitum): All animals were fed PMI Nutrition International, LLC Certified Rodent LabDiet 5002 ad libitum, except during the neurobehavioral evaluations and when fasted prior to sacrifice.
- Water (e.g. ad libitum): All animals were provided tap water ad libitum
- Acclimation period: Upon arrival at DuPont Haskell, all animals were housed in quarantine. The animals were:
• quarantined for 6 days.
• pair housed
• identified by a pretest animal number, which was tattooed on the animal’s tail.
• weighed during acclimation.
• observed with respect to weight gain and any gross signs of disease or injury.
• given an ophthalmology evaluation and an abbreviated neurobehavioral evaluation.
The animals were released from quarantine by the designee of the animal resources supervisor based on body weights and clinical signs.s.

DETAILS OF FOOD AND WATER QUALITY: As specified in the performing laboratory animal health and environmental monitoring program, the following procedures are performed periodically to ensure that contaminant levels are below those that would be expected to impact the scientific integrity of the study:
• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, and other contaminants.
• Samples from freshly washed cages and cage racks are analyzed to ensure adequate sanitation by the cagewashers.
Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional requirements and not to exceed stated maximum concentrations of key contaminants, including specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The presence of these contaminants below the maximum concentration stated by the manufacturer would not be expected to impact the integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-26ºC (68-79ºF)
- Humidity: 30-70%
- Photoperiod: 12 hour light/dark cycle.

IN-LIFE DATES: From July 24, 2014 to October, 24, 2014

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because it is a potential route of human exposure and is the most efficient way to deliver an accurate dose.

Vehicle:

water

Details on oral exposure:

The test substance was mixed with deionized water. Neither the amount nor nature of any contaminants in the vehicle affected the integrity or validity of this study. Dose formulations were prepared with a correction for the total amount of protein (mg)/mL. Dosing formulations of the test substance were prepared approximately weekly, stored refrigerated, and used within the established range of stability. Animals were dosed daily at approximately the same time (± 2 hours), except on days of neurobehavioral evaluations, by intragastric intubation at a dose volume of 10 mL/kg body weight for at least 90 days. The amount of test substance each animal received was based on the most recently collected body weight and the formulation concentration. Control animals were dosed with deionized water at a volume of 10 mL/kg of body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance was assumed to be a solution and therefore, homogeneity was not analyzed. For concentration verification, duplicate samples of each test dosing formulation were collected near the beginning and end of the study and were frozen at <-10ºC. For stability testing, duplicate samples of neat test substance and low and high dose formulations were collected near the middle of the study, stored refrigerated for 21 days, and then frozen at <-10ºC. Concentration samples were inadvertently not collected near the middle of the study, but the samples collected for stability near the middle of the study were sufficient to verify the concentration and stability of the low and high dose formulations at that time point. All samples were frozen at <-10ºC and shipped on dry ice to the sponsor analytical laboratory for analysis.

Samples were analyzed for stability and concentration of protein (mg/mL) by C/H/N/S Elemental Analysis using a nitrogen/protein analyzer. Two replicates of each sample were initially analyzed. Back-up samples collected near the beginning of the study (low and mid dose) and near the end of the study (high dose) were analyzed (one replicate each) to confirm the results of the initial analysis. The results reported are an average of the two or three replicates analyzed for each formulation.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 other: mg TP/kg bw/day

Remarks:

equivalent to 322.25 mg TOS/kg bw/day

Doses / concentrations 2

Dose / conc.:

500 other: mg TP/kg bw/day

Remarks:

equivalent to 644.5 mg TOS/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 other: mg TP/kg bw/day

Remarks:

equivalent to 1289 mg TOS/kg bw/day

No. of animals per sex per dose:

10 female and 10 male rats

Control animals:

yes, concurrent vehicle

Details on study design:

Three groups of young adult male and female rats (10/sex/group) were dosed by oral gavage for 91 days (males) or 92 days (females) with the test substance, diluted in deionized water, at doses of 250 (low dose), 500 (mid dose), or 1000 (high dose) mg total protein/kg bw/day. The control group was dosed with deionized water. All animals received weekly evaluations of bodyweight and nutritional parameters and clinical observations. Ophthalmology and neurobehavioral parameters were evaluated during the acclimation period (pretest) and near the end of the study. Clinical pathology parameters (hematology, coagulation, clinical chemistry, urinalysis), and anatomic pathology parameters (organ weights, gross and microscopic pathology) were evaluated at the end of the study. The concentrations and stability of the dosing formulations were verified under the conditions of the study. All animals survived to scheduled necropsy.

Examinations

Observations and examinations performed and frequency:

- Body Weights: All animals were weighed weekly, on the days of neurobehavioral evaluations, and on the days of sacrifice.
- Food Consumption and Food Efficiency: The amount of food consumed by each animal over each weighing interval was determined by weighing each feeder at the beginning and end of the interval and subtracting the final weight of the feeder during the interval from the initial weight. Cage food consumption was divided by the number of animals in the cage to calculate average individual animal food consumption. From these measurements, mean daily food consumption over the interval was determined. From the food consumption and body weight data, the mean daily food efficiency was calculated.
- Clinical Observations and Mortality:
1. Daily Animal Health Observations: Cage-site examinations to detect moribund or dead animals and abnormal behavior and/or appearance among animals were conducted at least twice daily throughout the study.
2. General Clinical Observations: An additional cage-site evaluation was conducted daily at approximately 2 hours (± 1 hour) post-dosing to detect acute clinical signs of systemic toxicity. An excursion from this time range occurred on one day, which did not impact the integrity of the study.
3. Detailed Clinical Observations: At every weighing (excluding weights on days of neurobehavioral evaluations and necropsy), each animal was individually handled and examined for abnormal behavior and appearance. Detailed clinical observations in a standardized arena were also evaluated on all animals. The detailed clinical observations included (but were not limited to) evaluation of fur, skin, eyes, mucous membranes, occurrence of secretions and excretions, autonomic nervous system activity (lacrimation, piloerection, and unusual respiratory pattern), changes in gait, posture, response to handling, presence of clonic, tonic, stereotypical, or bizarre behavior.
- Ophthalmology Evaluation: Two ophthalmology examinations were conducted by a veterinary ophthalmologist. The baseline examination was performed on all animals received for the study, prior to assignment to groups. All surviving animals were examined prior to the final sacrifice.
Both eyes of each animal were examined by focal illumination and indirect ophthalmoscopy. The eyes were examined in subdued light after mydriasis had been produced.
- Neurobehavioral Evaluation: Neurobehavioral evaluation, consisting of abbreviated functional observational battery (FOB) assessments and motor activity (MA), was conducted on all animals during acclimation baseline) and during week 13 on all surviving animals. For the baseline evaluation, FOB and MA assessments were also conducted on spare animals in the event that replacements were needed during acclimation or on test day 1. Animals were counterbalanced by sex and treatment and tested in replicates over multiple days to minimize the influence of uncontrolled factors. The experimenter conducting the FOB was blind with respect to the group designation of the animal. The testing was performed by the same person each time. FOB and MA evaluations were conducted in a sound-attenuated room equipped with a white-noise-generation system to minimize variations in environmental test conditions. Animals were acclimated at least for 10 minutes in the FOB laboratory prior to initiation of evaluation. Body weights were collected during the FOB assessment, but were not compared statistically.
- Clinical Pathology Evaluation: A clinical pathology evaluation was conducted on all surviving animals prior to the scheduled sacrifice. The day before collection of samples for the clinical pathology evaluation, the animals were placed in metabolism cages. These animals were fasted for at least 15 hours and urine was collected from each animal and analyzed on the day of collection. On the morning after the fast, blood samples for hematology and clinical chemistry measurements were collected via sublingual bleeding from each animal while under isoflurane anesthesia. Approximately 500 μL of blood was collected into a tube containing K2EDTA anticoagulant (ethylenediaminetetraacetic acid) for the hematology parameters and analyzed on the day of collection. Approximately 750 μL of blood was collected into a tube containing no anticoagulant for the serum clinical chemistry parameters. Samples in serum tubes were allowed to clot, centrifuged, and the resultant serum either analyzed on the day of collection or stored at <-60°C until analyzed. Blood samples for coagulation parameters were collected at sacrifice from the abdominal vena cava of each animal while the animal was under isoflurane anesthesia. Approximately 1.8 mL of blood was collected into a tube containing 3.2% sodium citrate anticoagulant and centrifuged; the resultant plasma was stored at <-60°C until analyzed for coagulation parameters. Additional blood collected from the vena cava was placed in a serum tube, allowed to clot, centrifuged, and the resultant serum was stored at <-60°C. Serum was discarded without analysis because further tests were not required to support experimental findings. Bone marrow smears were prepared at sacrifice from all surviving animals. Bone marrow smears were stained with Wright-Giemsa stain, but analysis was not necessary to support experimental findings. All blood samples were evaluated for quality by visual examination.

Sacrifice and pathology:

After at least 90 days on study (test days 92 and 93 for males and females, respectively), all rats survived to the scheduled study completion and were sacrificed and necropsied for evaluation of subchronic toxicity.

Rats sacrificed by design (scheduled sacrifice) were fasted at least 15 hours (with access to water) beginning on the afternoon before their scheduled necropsy. The order of sacrifice for scheduled deaths was stratified across groups within each sex. Rats were euthanized by exsanguination while under isoflurane anesthesia and a complete necropsy was performed on each rat. The necropsy included examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.

All tissues collected from rats in the control (Group 1; 0 mg/kg/day) and high-concentration (Group 4; 1,000 mg/kg/day) groups were trimmed, routinely processed, embedded in paraffin, sectioned approximately 5-6 microns thick, stained with hematoxylin and eosin (H&E), and prepared to slides for microscopic evaluation. Meaningful gross observations, from all rats on study, were also processed to slides and evaluated. Gross lesions for which microscopic examination would not be additive (e.g., osteoarthritis, pododermatitis, tail chronic dermatitis, calculus, and deformities of the teeth, toe, tail, or ear pinna) were not processed to slides. Since microscopic examination of the high-dose tissues did not reveal any test substance-related findings (i.e., no suspect target organs), additional tissues from the low and intermediate-dose groups were not prepared to slide and examined.

Statistics:

Significance was judged at p <0.05. Separate analyses were performed on the data collected for each sex.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

All rats survived until the scheduled terminal sacrifice. There were no clinical signs of toxicity attributed to test substance exposure.

One control male exhibited lung noise between test days 85 and 91, which was likely a response to the dosing procedure and did not impact the interpretation of the study. The only other clinical signs noted during the study were hair loss and scabs, which were distributed across treatment groups and are common background observations in rats of this strain and age.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on body weight or body weight gain in males or females administered up to 1000 mg/kg/day. Any statistical differences between treatment groups were considered to be unrelated to test substance exposure, as described below.
Body weight gain for test day 43-50 was lower (p<0.05) in males at 1000 mg/kg/day compared with controls. Body weight gain for test day 78-85 was higher (p<0.05) in males at 250 mg/kg/day compared with controls. These transient differences were not attributed to test substance exposure because there were no other statistically significant differences in body weight or body weight gain in either sex at any interval or time point, including the final body weight or overall (test day 1-91) body weight gain.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food consumption in males or females administered up to 1000 mg/kg/day. Any statistical differences between treatment groups were considered to be unrelated to test substance exposure, as described below.

Food consumption was higher (p<0.05) during two weekly intervals (test day 15-22 and 85-91) in males at 250 mg/kg/day and was lower (p<0.05) during a single weekly interval (test day 85-91) in females at 500 mg/kg/day, compared with controls. These differences were considered spurious for the following reasons: they were not consistent across sexes and did not occur in a dose-related manner; the higher or lower food consumption values corresponded to slightly higher or lower body weight gains, respectively (not statistically significant), such that there were no statistically significant differences in food efficiency during these weekly intervals; and there were no differences in overall food consumption or food efficiency in males or females at any dose level.

Food efficiency:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food efficiency in males or females administered up to 1000 mg/kg/day. Any statistical differences between treatment groups were considered to be unrelated to test substance exposure, as described below.

Food efficiency was lower or higher (p<0.05) during a single weekly interval each in males at 250 mg/kg/day, 500 mg/kg/day, and 1000 mg/kg/day, compared with controls. These transient differences were considered spurious because they were not reflected in statistically significant differences in overall food efficiency for males and there were no corroborative differences in weekly or overall food efficiency for females at any dose level.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related abnormalities noted during the ophthalmology evaluation in males or females at any exposure level. One low dose (250 mg/kg/day) female exhibited diffuse retinal degeneration in the left eye, which is a common background observation in rats of this strain and age.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no statistically significant changes in hematology parameters in male or female animals.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no statistically significant changes in clinical chemistry parameters in male or female animals.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no statistically significant changes in urinalysis parameters in male or female animals.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test substance-related terminal body weight or organ weight effects. There were no statistically significant organ weight changes in absolute, relative to brain or relative to body weight values for females. The single statistically significant organ weight difference in males was a decrease in the spleen (relative to body weight) at 500 mg/kg/day. This difference was not test substance related as it was: not dose dependent (only observed at mid-dose); no similar finding was present in female spleen weights; there was no microscopic correlate. All differences from controls for absolute, relative to brain and relative to body weight values were considered to be spurious and unrelated to test substance administration.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related gross observations in males or females. All gross observations were background findings in rats of this age and strain.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

other: TP (Total Protein)

Sex:

male/female

Basis for effect level:

other: lack of test substance-related effects at the highest dose tested

2

Key result

Dose descriptor:

NOAEL

Effect level:

1 289 mg/kg bw/day (nominal)

Based on:

other: TOS (Total Organic Solids)

Sex:

male/female

Basis for effect level:

other: lack of test substance-related effects at the highest dose tested

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the NOAEL for the test substance was 1000 mg TP/kg bw/day for male and female rats, based on a lack of test substance-related effects at the highest dose tested. This NOAEL is equivalent to 1289 mg TOS/kg bw/day in males and females.

Executive summary:

The study was conducted to evaluate the sub-chronic toxicity of Catalase when administered by oral gavage to male and female rats for at least 90 days. The study was conducted in accordance with the OECD Guideline 408 in compliance with GLP.

 

Three groups of young adult male and female Crl:CD(SD) rats (10/sex/group) were dosed by oral gavage for 91 days (males) or 92 days (females) with the test substance, diluted in deionized water, at doses of 250 (low dose), 500 (mid dose), or 1000 (high dose) mg TP/kg bw/day. The control group was dosed with deionized water. All animals received weekly evaluations of bodyweight and nutritional parameters and clinical observations. Ophthalmology and neurobehavioral parameters were evaluated during the acclimation period (pretest) and near the end of the study. Clinical pathology parameters (hematology, coagulation, clinical chemistry, urinalysis), and anatomic pathology parameters (organ weights, gross and microscopic pathology) were evaluated at the end of the study. The concentrations and stability of the dosing formulations were verified under the conditions of the study.

 

All animals survived to scheduled necropsy. No test substance-related effects were observed on body weight or nutritional parameters, clinical or ophthalmology observations, neurobehavioral parameters, clinical pathology parameters, or anatomic pathology parameters.

 

Under the conditions of this study, the NOAEL for the test substance was 1000 mg TP/kg bw/day for male and female rats, based on a lack of test substance-related effects at the highest dose tested. This NOAEL is equivalent to 1289 mg TOS/kg bw/day in males and females.