Subtilisin

Endpoint summary

Description of key information

The repeated dose oral toxicity of subtilisin has been tested in a 90-day study, the repeated dose dermal toxicity was tested in a 28-day study, while the repeated dose inhalation toxicity was waived.
- The repeated dose oral toxicity was a subchronic toxicity test similar to the principles of OECD guideline 408 (version 25 june 2018), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the highest dose level administered, equivalent to 572 mg enzyme concentrate dry matter/kg bwt/day (equivalent to 302.6 mg active enzyme protein/kg bwt/day).
- The dermal study concluded that the dose applied daily, 10 mg/kg/day (only one dose level tested), was without any significant effects, only sporadic, minimal grade local skin reactions. It is further highly unlikely that enzymes should be absorbed through the skin due to the molecular weight and the physico-chemical properties of the protein molecule.
- The inhalation study was waived because exposure is too low to exert any toxicological concern. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitization 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:

20 November 2018 - 29 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25 June 2018

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: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 43 to 49 days
- Weight at study initiation: Males: 133-183 g, Females: 120-157 G
- Fasting period before study: None
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at
appropriate intervals.
- Diet (e.g. ad libitum): Teklad 2014C Diet ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not
recirculated.
- Photoperiod: 12 hrs light/12 hrs dark

IN-LIFE DATES: From: 5 December 2018 To: 7 March 2019

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Reverse osmosis water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 57.2, 188.8 and 572.0 mg enzyme concentrate dry matter/kg bodyweight
- Amount of vehicle (if gavage): 5 mL/kg body weight

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

57.2 mg/kg bw/day (nominal)

Remarks:

enzyme concentrate dry matter

Dose / conc.:

188.8 mg/kg bw/day (nominal)

Remarks:

enzyme concentrate dry matter

Dose / conc.:

572 mg/kg bw/day (nominal)

Remarks:

enzyme concentrate dry matter

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Fasting period before blood sampling for clinical biochemistry: Food removed overnight

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the
occupants.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: One week before treatment, on the day that the treatment commenced, once a week throughout the study and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION: Fluid intake was assessed by daily visual observation. No significant effect was observed and, consequently, quantitative measurements were not performed

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pretreatment and week 12
- Dose groups that were examined: all animals at pretreatment and control group and highest dose group at week 12.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
Hematocrit (Hct), Hemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH)*, Mean cell hemoglobin concentration (MCHC)*, Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC)

Differential leucocyte count:
Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 13
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked:
Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Bile acids (Bi Ac), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Cholesterol (HDL), Cholesterol (LDL), sodium (Na), Potassium (K), Chloride (Cl), Total protein (Total Prot), Albumin (Alb)

PLASMA/SERUM HORMONES: Yes
- Time of blood sample collection: At termination
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked:
Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
Estrus cycles: Wet smears were taken from the vagina of all females using pipette lavage for 4 days before scheduled necropsy. Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus) and were used to assist in the histological evaluation of estrogen sensitive tissues.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A male animal in the highest dose group died in week 11. Lung lesions were considered the major contributing factor to death and were most likely caused by reflux and/or aspiration of dose. Consequently, this death was attributed to the dose administration procedure and not to toxicity.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

No effects on estrus cycle.

Details on results:

HEMATOLOGY, PERIPHERAL BLOOD: All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation.
CLINICAL CHEMISTRY: All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation.
THYROID HORMONE ANALYSIS: The TSH concentrations were statistically significantly higher
than those of the controls for all groups of treated males but there was no dose response and
females were not similarly affected.

Key result

Dose descriptor:

NOAEL

Effect level:

572 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 at a dose with 100% of the test material.

Key result

Critical effects observed:

not specified

Conclusions:

It is concluded that daily oral administration of Subtilisin, batch PPA55402 to Han Wistar
rats at dose levels up to 100% of the test batch for 13 weeks was well-tolerated, with no
evidence of any adverse finding at any of the administered doses. Consequently, the
no-observed-adverse-effect level (NOAEL) was considered to be 572 mg enzyme concentrate dry matter/kg bwt/day (equivalent to 302.6 mg active enzyme protein/kg bwt/day).

Executive summary:

The purpose of this study was to assess the systemic toxic potential of Subtilisin,
batch PPA55402, an enzyme intended for use in the feed or food industry, when administered orally (by gavage) to Han Wistar rats for 13 weeks. Three groups, each comprising 10 male and 10 female rats received doses of 10, 33 or 100% of Subtilisin, batch PPA55402 (equivalent to 57.2, 188.8 or 572.0 mg enzyme concentrate dry matter/kg bwt/day, respectively). A similarly constituted control group received the vehicle (reverse osmosis water) at the same volume dose (5 mL/kg bwt/day).
During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, visual water consumption, ophthalmoscopy, hematology (peripheral blood), blood chemistry, estrous cycle, thyroid hormone, organ weight, macropathology and histopathology investigations were undertaken.

The general appearance and behaviour of the animals and sensory activity, grip strength and motor activity were unaffected by treatment. There was also no effect of treatment on body weight gain or food and water consumption. One high dose male died in Week 11 after suspected aspiration of dose and, consequently, the death of this animal was attributed to the dose administration procedure.
There were no treatment-related ophthalmoscopic findings. The haematological and blood chemistry investigation did not indicate any toxicologically significant findings. Estrous cycles at the end of the treatment period were unaffected.
Serum triiodothyronine (T3) and thyroxine (T4) concentrations were unaffected by treatment. Serum thyroid stimulating hormone (TSH) concentrations were higher than those of the controls at all dose levels in males but there was no dose response and females were not similarly affected. Consequently, the variations reported for TSH were, in the absence of any physiological response, considered to represent normal biological variation and were of no toxicological importance.
Organ weights were unaffected and there were no treatment-related macroscopic or histopathological findings.

It is concluded that daily oral administration of Subtilisin, batch PPA55402 to Han Wistar rats at dose levels up to 100% of the test batch for 13 weeks was well-tolerated, with no evidence of any adverse finding at any of the administered doses. Consequently, the no-observed-adverse-effect level (NOAEL) was considered to be 572 mg enzyme concentrate dry matter/kg bwt/day
(equivalent to 302.6 mg active enzyme protein/kg bwt/day).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

572 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

High quality. The key study is GLP compliant and Klimisch 1.

Repeated dose toxicity: inhalation - systemic effects

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:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The subacute percutaneous toxicity of Subtilisin was assessed. The systemic and local effects of daily repeated applications of 10 mg/kg/day of the test material Subtilisin, prepared as a 0.5% w/v solution in water and 0.5% w/v in sodium tripolyphosphate buffer, respectively. The application was performed daily for 28 consecutive days without occlusion to the abraded and intact skin of the albino rabbit, an area equal to 10% of the total body surface clipped free of hair. Four female and four male rabbits were used per group, i.e. 32 rabbits in total including two negative control groups.

GLP compliance:

no

Remarks:

The study was performed before GLP was implemented but was performed according to state of the art at that time.

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Morton Commercial Rabbits, Parsonage Farm, Stanstedt, UK
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 2.2 - 2.8 kg
- Age at study initiation: Young adults, 3 - 3.5 months
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 14-18C
- Humidity : 50% ( range 40-60 %)
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs

Type of coverage:

open

Vehicle:

other: water, respectively sodium tripolyphosphate buffer

Details on exposure:

TEST SITE
- Area of exposure: 10% of body surface
- Time intervals for shavings or clippings: The animals were shaven as needed - no specific interval given in report.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Four hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg
- Concentration (if solution): 0.5% w/v
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0.1% sodium tripolyphosphate buffer

USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Four hours per day

Frequency of treatment:

Each day for 28 days.

Remarks:

Doses / Concentrations:
10 mg/kg, i.e. 2.0 mL/kg of a 0.5% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal per unit body weight
8.8 mg enzyme concentrate dry matter/kg

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on experience from short term dose-range studies, the dose was selected to provide some toxicological effects but still avoiding extreme irritation as an endpoint.

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Days 1, 2, 3, 7, 14, 21, 28

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals (32)
- Parameters checked: hemoglobin concentration (Hb), Erythrocyte count (RBC), Leucocyte count, total (WBC), neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and packed cell volume (PCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: All animals (32)
- Parameters checked: Urea concentration, glucose concentration, total protein concentration, electrophoretic protein fractions, alkaline phosphatase activity (SAP), glutamate-pyruvate transaminase activity (SGPT), glutamate-oxalacetate transaminase activity (SGOT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes ( Appendix 9 in report)
HISTOPATHOLOGY: Yes (Appendix 9 in report)

Other examinations:

Twelve main organs were weighed

Statistics:

Organ weights were evaluated by analysis of variance.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

skin reactions were confined to sporadic, minimal grade. The observed responses were considered to be related to the daily mechanical handling and material application.

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

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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

Description (incidence and severity):

Any effects were confined to slight acanthosis, and a variable, slight infiltration of the underlying dermis by leucocytes

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical effects and no mortality

BODY WEIGHT AND WEIGHT GAIN: Within normal limits during the study.

FOOD CONSUMPTION: Within normal ranges during the study.

HAEMATOLOGY: Within normal ranges, unaffected by treatment.

CLINICAL CHEMISTRY Within normal ranges, unaffected by treatment.

NEUROBEHAVIOUR: Behaviour normal throughout the study

ORGAN WEIGHTS: No differences between groups.

GROSS PATHOLOGY: No treatment related gross lesions present

HISTOPATHOLOGY: NON-NEOPLASTIC There were no treatment related effects other than minor local skin effects. These were slight acanthosis, occasional patchy parakeratosis and variable slight infiltration of underlying dermis by leucocytes.

Key result

Dose descriptor:

conc. level:

Effect level:

> 1.4 mg/kg bw/day (nominal)

Based on:

dissolved

Remarks:

0.5% w/v solution in water or buffer

Sex:

male/female

Basis for effect level:

other: detailed skin reactions, clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Critical effects observed:

not specified

Conclusions:

The only effects of 28-day repeated treatment of intact and abraded skin with Subtilisin, buffered and unbuffered, were microscopically detected minor changes at the site of application.

Executive summary:

A percutaneous 28-day repeated application study in rabbits was conducted by Life Science Research (now Huntingdon Life Sciences Ltd.) to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause dermal toxicity. Only one dose was applied daily, 10 mg/kg/day, diluted in either water or buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each group consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed.

The study concluded that the test substance, Subtilisin, was without any significant effects and thus may be considered virtually harmless.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimisch 2 study - only one dose level tested.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Critical effects observed:

not specified

Reference 2

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The subacute percutaneous toxicity of Subtilisin was assessed. The systemic and local effects of daily repeated applications of 10 mg/kg/day of the test material Subtilisin, prepared as a 0.5% w/v solution in water and 0.5% w/v in sodium tripolyphosphate buffer, respectively. The application was performed daily for 28 consecutive days without occlusion to the abraded and intact skin of the albino rabbit, an area equal to 10% of the total body surface clipped free of hair. Four female and four male rabbits were used per group, i.e. 32 rabbits in total including two negative control groups.

GLP compliance:

no

Remarks:

The study was performed before GLP was implemented but was performed according to state of the art at that time.

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Morton Commercial Rabbits, Parsonage Farm, Stanstedt, UK
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 2.2 - 2.8 kg
- Age at study initiation: Young adults, 3 - 3.5 months
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 14-18C
- Humidity : 50% ( range 40-60 %)
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 10 hrs/14 hrs

Type of coverage:

open

Vehicle:

other: water, respectively sodium tripolyphosphate buffer

Details on exposure:

TEST SITE
- Area of exposure: 10% of body surface
- Time intervals for shavings or clippings: The animals were shaven as needed - no specific interval given in report.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: Four hours

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg/kg
- Concentration (if solution): 0.5% w/v
- Constant volume or concentration used: yes
- For solids, paste formed: no

VEHICLE
- Amount(s) applied (volume or weight with unit): 2 mL/kg
- Concentration (if solution): 0.1% sodium tripolyphosphate buffer

USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Four hours per day

Frequency of treatment:

Each day for 28 days.

Remarks:

Doses / Concentrations:
10 mg/kg, i.e. 2.0 mL/kg of a 0.5% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal per unit body weight
8.8 mg enzyme concentrate dry matter/kg

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on experience from short term dose-range studies, the dose was selected to provide some toxicological effects but still avoiding extreme irritation as an endpoint.

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Days 1, 2, 3, 7, 14, 21, 28

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: all animals (32)
- Parameters checked: hemoglobin concentration (Hb), Erythrocyte count (RBC), Leucocyte count, total (WBC), neutrophils (N), lymphocytes (L), eosinophils (E), basophils (B), monocytes (M) and packed cell volume (PCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: All animals (32)
- Parameters checked: Urea concentration, glucose concentration, total protein concentration, electrophoretic protein fractions, alkaline phosphatase activity (SAP), glutamate-pyruvate transaminase activity (SGPT), glutamate-oxalacetate transaminase activity (SGOT)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes ( Appendix 9 in report)
HISTOPATHOLOGY: Yes (Appendix 9 in report)

Other examinations:

Twelve main organs were weighed

Statistics:

Organ weights were evaluated by analysis of variance.

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Description (incidence and severity):

skin reactions were confined to sporadic, minimal grade. The observed responses were considered to be related to the daily mechanical handling and material application.

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

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

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

Description (incidence and severity):

Any effects were confined to slight acanthosis, and a variable, slight infiltration of the underlying dermis by leucocytes

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: No clinical effects and no mortality

BODY WEIGHT AND WEIGHT GAIN: Within normal limits during the study.

FOOD CONSUMPTION: Within normal ranges during the study.

HAEMATOLOGY: Within normal ranges, unaffected by treatment.

CLINICAL CHEMISTRY Within normal ranges, unaffected by treatment.

NEUROBEHAVIOUR: Behaviour normal throughout the study

ORGAN WEIGHTS: No differences between groups.

GROSS PATHOLOGY: No treatment related gross lesions present

HISTOPATHOLOGY: NON-NEOPLASTIC There were no treatment related effects other than minor local skin effects. These were slight acanthosis, occasional patchy parakeratosis and variable slight infiltration of underlying dermis by leucocytes.

Key result

Dose descriptor:

conc. level:

Effect level:

> 1.4 mg/kg bw/day (nominal)

Based on:

dissolved

Remarks:

0.5% w/v solution in water or buffer

Sex:

male/female

Basis for effect level:

other: detailed skin reactions, clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology;

Critical effects observed:

not specified

Conclusions:

The only effects of 28-day repeated treatment of intact and abraded skin with Subtilisin, buffered and unbuffered, were microscopically detected minor changes at the site of application.

Executive summary:

A percutaneous 28-day repeated application study in rabbits was conducted by Life Science Research (now Huntingdon Life Sciences Ltd.) to assess the potential of the test substance, Subtilisin (batch FPF 312/20), to cause dermal toxicity. Only one dose was applied daily, 10 mg/kg/day, diluted in either water or buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each group consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles was the same and state of the art was followed.

The study concluded that the test substance, Subtilisin, was without any significant effects and thus may be considered virtually harmless.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

167 µg/cm²

Study duration:

subacute

Species:

rabbit

Quality of whole database:

Klimisch 2 study - only one dose level tested.

Additional information

The repeated dose oral toxicity of subtilisin has been tested in a 90-day study, the repeated dose dermal toxicity was tested in a 28-day study, while the repeated dose inhalation toxicity was waived.

- The repeated dose oral toxicity was a subchronic toxicity test similar to the principles of OECD guideline 408 (version 25 June 2018), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was considered to be the intermediate dose level administered, equivalent to 572 mg enzyme concentrate dry matter/kg bwt/day (equivalent to 302.6 mg active enzyme protein/kg bwt/day).

- The dermal study concluded that the dose applied daily, 10 mg/kg/day (only one dose tested), was without any significant effects, only sporadic, minimal grade local skin reactions. It is further highly unlikely that enzymes should be absorbed through the skin due to the molecular weight and the physico-chemical properties of the protein molecule.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitization by inhalation. Therefore, based on weight of evidence, subtilisin does not exert any repeated dose inhalation toxicity to workers or consumers.

Justification for classification or non-classification

No classification as NOAEL is above guidance level of 100 mg/kg bw/day. The installed controls to insure no risk of respiratory sensitization generally ensure no repeated dose inhalation or dermal toxicity to workers or consumers