Registration Dossier

Administrative data

Description of key information

The repeated dose oral and dermal toxicity of alpha-amylase has been tested, while the repeated dose inhalation toxicity was waived.

- The dermal study was a one-dose study, dose being 2.0 mL/kg of a 0.62% w/v solution, i.e. 12.4 mg/kg, in water or buffer, respectively, corresponding to a dose of 6 KNU/kg/day expressed in alpha-amylase activity. This gave only mild local skin reactions. This outcome is supported by the knowledge of 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, 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.

- 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 the undiluted alpha-amylase batch/kg bw/day or 1100 mg Total Organic Solids (TOS)/kg bw/day.

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

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 June 2009 - 20 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
revised 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK, Ltd
- Age at study initiation: 43 days
- Weight at study initiation: 176-221 g for males; 150-189 g for females
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C (on one occasion during week 13 of treatment a minimum temperature of 17°C was recorded but this minor deviation from target was considered to have no effect on the health and welfare of the animals and no effect on the results of the study)
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2009-07-01 To: 2009-10-09
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 33 and 100%, corresponding to 110, 362 and 1100 mg total organic solids (TOS) per kg body weight.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Water for formulation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP.
Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken once during weeks 1, 6 and 13. No significant differences were found between weeks and no significant differences were found between the achieved concentration for the high dose group and the 100% undiluted tox-batch, demonstrating satisfactory formulation.

Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
1 other: mL/kg bw/day
Remarks:
corresponding to 0.110 g TOS/kg/day
Dose / conc.:
3.3 other: mL/kg bw/day
Remarks:
corresponding to 0.362 g TOS/kg bw/day
Dose / conc.:
10 other: mL/kg bw/day
Remarks:
corresponding to 1.10 g TOS/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected on the basis of results from studies performed on other similar enzyme preparations. The highest dose (10 mL/kg/day) was the maximum practical dose and represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg body weight, which is the maximum practical volume-dose for repeat dose oral administration. The lower doses were selected using an approximate ratio of 3.3 between doses.


Positive control:
Not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 and weekly thereafter, detailed observations were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Daily by visual observation

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 12
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (Plt)
Prothrombim time (PT)
Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)
Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full macroscopic examination
HISTOPATHOLOGY: Yes. Adrenals, brain, femur with joint, heart, kidneys, liver, lungs, spinal cord, sternum, stomach, thyroid and uterus were preserved and for the control group and the high dose group these tissues were examined.
Other examinations:
FAECAL ANALYSIS: No

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy
Statistics:
Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, organ weights and any data derived from these was performed. The level of probability chosen as significant was p<0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
One death of a high dose female was clearly accidental.
Mortality:
no mortality observed
Description (incidence):
One death of a high dose female was clearly accidental.
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):
Small increase of lymphocyte numbers at high dose in both sexes. No histopath evidence of inflammatory changes in any tissue. Compared to background data for lymphocyte counts the study values were not abnormal. Considered of no toxicological significance
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Small increase of plasma potassium concentration in high dose males. No evidence of any effect upon the kidneys or water balance and no similar finding in females. The change was therefore considered of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Slightly lower spleen weight in high dose females. Males not affected. No histopathological evidence of inflammatory changes in any tissue and no evidence of immunosuppression. The finding was therefore considered to be of no toxicological significance.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
HAEMATOLOGY AND ORGAN WEIGHTS: A small increase of lymphocyte numbers in the peripheral blood was reported at high dose (1100 mg TOS/kg body weight/day) in both sexes, but particularly for the females in which there was also a small reduction of spleen weight. There was no evidence at the histopathological examination for any chronic inflammatory change in any tissue and immunosuppression was discounted because this would result in low (not high) lymphocyte counts. Review of the findings against the background control data for lymphocyte counts also demonstrated that the study values were not abnormal. These findings were, therefore, considered not to be of any toxicological significance and there was no evidence that alpha-amylase, PPY 29933 had any adverse effect on the immune system.


Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 100 mg/kg bw/day (nominal)
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Key result
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) in rats was 10 mL of the undiluted alpha-amylase/kg body weight/day equivalent to 1100 mg Total Organic Solids (TOS)/kg body weight/day.
Executive summary:

The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 408 (revised 1998), and in compliance with GLP. The purpose of the study was to satisfy regulatory demands because the enzyme is used for production of food in EU.

In conclusion, oral administration (by gavage) of alpha-amylase to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted test material/kg body weight/day or 1100 mg Total Organic Solids (TOS)/kg body weight/day for thirteen weeks was well-tolerated and did not produce any toxicologically significant changes.

Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL alpha-amylase /kg body weight/day or 1100 mg Total Organic Solids (TOS)/kg body weight/day.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 1996 - 15 November 1996
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Ltd., Germany
- Age at study initiation: 37-41 days
- Weight at study initiation: 172-197 g for males; 119-142 g for females
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet : Standard pelleted rodent diet ad libitum
- Water : Tap water ad libitum
- Acclimatization period: At least 10 days

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C (on one occasion during c. 44 hours in total on days 21-23 of treatment the temperature was above 25°C reaching a maximum of 26.5°C during a period of c. 3 hours on day 22. This deviation from target was considered to have no effect on the health and welfare of the animals and no effect on the results of the study)
- Humidity : 35-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2009-07-01 To: 2009-10-09
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 10, 50 and 100%, corresponding to 132, 660 and 1320 mg total organic solids (TOS) per kg body weight.
- Amount of vehicle (if gavage): constant volume 10 mL/kg body weight.
- Purity: Water for formulation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP.
Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken on day 0 and on day 27. No significant differences were found between sampling days and no significant differences were found between the achieved concentration for the high dose group and the 100% undiluted tox-batch, demonstrating satisfactory formulation.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Dose / conc.:
1 other: mL/kg bw/day
Remarks:
corresponding to 0.132 g TOS/kg bw/day
Dose / conc.:
5 other: mL/kg bw/day
Remarks:
corresponding to 0.66 g TOS/kg bw/day
Dose / conc.:
10 other: mL/kg bw/day
Remarks:
corresponding to 1.32 g TOS/kg bw/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected on the basis of results from studies performed on other similar enzyme preparations. The highest dose (10 mL/kg/day) was the maximum practical dose and represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg body weight, which is the maximum practical volume-dose for repeat dose oral administration.


Positive control:
Not included
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily on work days, once daily in weekends

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once weekly, detailed observations were recorded

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 4, 7, 11, 14, 18, 21, 25, 27 and 28 (and on day -3 to enable the allocation of the rats)

FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : No

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy week 4
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Haemoglobin
Packed cell volume
Erythrocyte count
Reticulocytes (blood smears were preparedand stained but not examined because there were no treatment related changes in red blood cell variables)
Total white blood cell count
Differential white blood cell count (in blood smears)
Prothrombin time
Thrombocyte count
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy week 4
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alanine aminotransferase activity (ALAT)
Aspartate aminotransferase activity(ASAT)
Gamma-glutamyl transferase activity (GGT)
Total bilirubin
Urea
Creatinine
Glucose
Cholesterol
Triglycerides
Phospholipids
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (C)
Inorganic phosphate
Total protein
Albumin
Ratio albumin to globulin

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 4
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : yes

Sacrifice and pathology:
GROSS PATHOLOGY: Yes, full macroscopic examination
HISTOPATHOLOGY: Yes. Adrenals, bone marrow (sternum), brain, heart, kidneys, liver, lungs, lymph nodes (mesenteric and axillary), ovaries, peripheral (sciatic) nerve, prostate, GALT (Peyers patches), gross lesions, small and large intestines, stomach, spinal cord, spleen, testes, thymus, thyroid, trachea and lung, urinary bladder and uterus were preserved and for the control group and the high dose group these tissues were examined.
Other examinations:
FAECAL ANALYSIS: No

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy
Statistics:
Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, organ weights and any data derived from these was performed. The level of probability chosen as significant was p<0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
One death of a high dose male due to a dosing error.
Mortality:
no mortality observed
Description (incidence):
One death of a high dose male due to a dosing error.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Changes in growth rate in high dose group were in opposite direction for males and females and were only slight and generally not statistically significant. Similar decreases were observed in males of the low-dose group without a dose-response relation.
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):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Slight decreased aspartate aminotransferase (ASAT) in high-dose females.
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not specified
Details on results:
BODY WEIGHT AND BODY WEIGHT GAIN: The changes in growth rate in high dose group (1320 mg TOS/kg body weight/day) were in opposite direction for males and females and were only slight and generally not statistically significant. Moreover, decreases of similar magnitude were observed in males of the low-dose group without a dose-response relation. Therefore, these fluctuations in growth rate are considered to be fortuitous findings.

CLINICAL CHEMISTRY: The decrease in ASAT activity in females of the high-dose group (1320 mg TOS/kg body weight/day) was only slight and not accompanied by changes in related clinical chemistry parameters or by effects on weight or pathology of the liver. Since, moreover an increase rather than a decrease in ASAT activity is considered the reflection of a toxic effect, this change is not ascribed to the treatment and is considered not to be of any toxicological significance.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 320 mg/kg bw/day (nominal)
Based on:
other: total organic solids (TOS)
Sex:
male/female
Basis for effect level:
other: NOAEL = Highest dose tested. In the absence of any toxicologically significant findings during the 4 week treatment period, the NOAEL in this study was considered to be the highest dose administered, 1320 mg TOS/kg body weight/day.
Key result
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) in rats was 10 mL of the undiluted alpha-amylase batch/kg body weight/day, equivalent to 1320 mg Total Organic Solids (TOS)/kg body weight/day.
Executive summary:

The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 407 (adopted July 1995), and in compliance with GLP.

In conclusion, oral administration (by gavage) of alpha-amylase to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted alpha-amylase batch/kg body weight/day or 1320 mg Total Organic Solids (TOS)/kg body weight/day for four weeks was well-tolerated and did not produce any toxicologically significant changes.

Consequently, the No Observed Adverse Effect Level (NOAEL) was considered to be the highest dose level administered, equivalent to 10 mL alpha-amylase/kg body weight/day or 1320 mg Total Organic Solids (TOS)/kg body weight/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 100 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:
Critical effects observed:
not specified
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

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The subacute percutaneous toxicity of Alpha-Amylase was assessed. The systemic and local effects of daily repeated applications of 6 KNU (Alpha-Amylase enzyme activity units)/kg/day of the test material, prepared as a 0.62% w/v solution in water and 0.62% 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 and environmental conditions:
- Source: Outbred New Zealand White rabbits, breeder not listed in report
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 1.97 - 2.84 kg
- Age at study initiation: Young adults, 2.5 - 3 months
- Diet (e.g. ad libitum): Standard diet (Diet RAF from Labsure Animal Foods, Poole, Dorset) ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 13-17
- 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 clipplings: 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): 2 mL/kg/day, corresponding to an amylase activity of 6 KNU/kg/day
- Concentration (if solution): 0.62% 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): 0.3 % sodium tripolyphosphate buffer


USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application to prevent ingestion of any test material.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Four hours per day
Frequency of treatment:
Each day for 28 days.
Dose / conc.:
12.4 other: mg/kg bw
Remarks:
Doses / Concentrations:
12.4 mg/kg, i.e. 2.0 mL/kg of a 0.62% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on experience, the dose was selected to avoid 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: Daily, just before dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment period

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined at weekly intervals throughout the treatment period and mean daily diet consumption calculated as g food/kg body weight/day.

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: 32
- The following parameters were examined: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- The following parameters were examined: urea, glucose, albumin, total protein, electrophoretic protein fractions, alkaline phosphatase, glutamate-pyruvate transaminase activity, glutamate-oxalacetate transaminase activity

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
HISTOPATHOLOGY: Yes (all animals - 20 organs/tissues examined in all groups)
Other examinations:
Eleven main organs were weighed
Statistics:
Organ weights were evaluated by analysis of variance.
Clinical signs:
no effects observed
Description (incidence and severity):
Two animals died during the study from unrelated causes as determined at necrosis
Dermal irritation:
no effects observed
Description (incidence and severity):
Skin reactions were confined to erythematous responses at the site of application, however no oedema.
Mortality:
no mortality observed
Description (incidence):
Two animals died during the study from unrelated causes as determined at necrosis
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):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Any effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No clinical effects. Two animals died during the study from unrelated causes as determined at necrosis.

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 effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.



Key result
Dose descriptor:
conc. level:
Effect level:
> 12.4 mg/kg bw/day (nominal)
Based on:
dissolved
Remarks:
0.62% 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;
Conclusions:
The only effects of 28-day repeated treatment of intact and abraded skin with alpha-amylase, buffered and unbuffered, were minor effects locally at the site of application without any initiation of any detectable systemic response. The local skin effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin. The local irritation effects could easily be caused by the content of smaller amounts of protease enzyme activity in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such side activities.
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, alpha-amylase (batch ATE 020), to cause dermal toxicity. Only one dose was applied daily, 12.4 mg/kg/day, diluted in water respectively buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each of the four groups applied consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles were the same and state of the art was followed. The study concluded that the test substance, alpha-amylase, was with only minor effects locally at the site of application without initiation of any systemic effects. The local effects seen at the site of application could easily be caused by the content of smaller amounts of protease enzyme activity in the test material, which was in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such protease side activities.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
12.4 mg/kg bw/day
Study duration:
subacute
Species:
rabbit

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Critical effects observed:
not specified
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The subacute percutaneous toxicity of Alpha-Amylase was assessed. The systemic and local effects of daily repeated applications of 6 KNU (Alpha-Amylase enzyme activity units)/kg/day of the test material, prepared as a 0.62% w/v solution in water and 0.62% 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 and environmental conditions:
- Source: Outbred New Zealand White rabbits, breeder not listed in report
- Fasting period before study: None
- Housing: individually in stainless steel cages
- Weight at study initiation: between 1.97 - 2.84 kg
- Age at study initiation: Young adults, 2.5 - 3 months
- Diet (e.g. ad libitum): Standard diet (Diet RAF from Labsure Animal Foods, Poole, Dorset) ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: minimum 14 days
- Temperature (°C): 13-17
- 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 clipplings: 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): 2 mL/kg/day, corresponding to an amylase activity of 6 KNU/kg/day
- Concentration (if solution): 0.62% 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): 0.3 % sodium tripolyphosphate buffer


USE OF RESTRAINERS FOR PREVENTING INGESTION: no - however, collars were worn by the animals four hours post-application to prevent ingestion of any test material.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Four hours per day
Frequency of treatment:
Each day for 28 days.
Dose / conc.:
12.4 other: mg/kg bw
Remarks:
Doses / Concentrations:
12.4 mg/kg, i.e. 2.0 mL/kg of a 0.62% w/v solution in water and sodium tripolyphosphate buffer, respectively.
Basis:
nominal
No. of animals per sex per dose:
4
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on experience, the dose was selected to avoid 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: Daily, just before dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly throughout the treatment period

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined at weekly intervals throughout the treatment period and mean daily diet consumption calculated as g food/kg body weight/day.

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: 32
- The following parameters were examined: haematocrit, haemoglobin concentration, erythrocyte count, total and differential leucocyte count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before commencement and after 4 weeks of treatment
- Animals fasted: No
- How many animals: 32
- The following parameters were examined: urea, glucose, albumin, total protein, electrophoretic protein fractions, alkaline phosphatase, glutamate-pyruvate transaminase activity, glutamate-oxalacetate transaminase activity

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
HISTOPATHOLOGY: Yes (all animals - 20 organs/tissues examined in all groups)
Other examinations:
Eleven main organs were weighed
Statistics:
Organ weights were evaluated by analysis of variance.
Clinical signs:
no effects observed
Description (incidence and severity):
Two animals died during the study from unrelated causes as determined at necrosis
Dermal irritation:
no effects observed
Description (incidence and severity):
Skin reactions were confined to erythematous responses at the site of application, however no oedema.
Mortality:
no mortality observed
Description (incidence):
Two animals died during the study from unrelated causes as determined at necrosis
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):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Any effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: No clinical effects. Two animals died during the study from unrelated causes as determined at necrosis.

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 effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin.



Key result
Dose descriptor:
conc. level:
Effect level:
> 12.4 mg/kg bw/day (nominal)
Based on:
dissolved
Remarks:
0.62% 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;
Conclusions:
The only effects of 28-day repeated treatment of intact and abraded skin with alpha-amylase, buffered and unbuffered, were minor effects locally at the site of application without any initiation of any detectable systemic response. The local skin effects were mild and confined to slight acanthosis, and very slight superficial dermatitis with no differentiation in response between intact and abraded skin. The local irritation effects could easily be caused by the content of smaller amounts of protease enzyme activity in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such side activities.
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, alpha-amylase (batch ATE 020), to cause dermal toxicity. Only one dose was applied daily, 12.4 mg/kg/day, diluted in water respectively buffer, to the closely-clipped dorsa of New Zealand White rabbits, equal to 10% of the body surface. Vehicle controls were included. Each of the four groups applied consisted of 4 males and 4 females. The study was conducted before GLP was implemented but the principles were the same and state of the art was followed. The study concluded that the test substance, alpha-amylase, was with only minor effects locally at the site of application without initiation of any systemic effects. The local effects seen at the site of application could easily be caused by the content of smaller amounts of protease enzyme activity in the test material, which was in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such protease side activities.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

The repeated dose oral and dermal toxicity of alpha-amylase has been tested, while the repeated dose inhalation toxicity were waived.

- The dermal study was a single-dose study, dose being 2.0 mL/kg of a 0.62% w/v solution, i.e. 12.4 mg/kg, in water or buffer, respectively, corresponding to a dose of 6 KNU/kg/day expressed in alpha-amylase activity. This gave only mild local skin reactions, most likely due to the content of smaller amounts of protease enzyme activity in addition to the main alpha-amylase activity. Alpha-amylase products of today do not contain such side activities. The overall conclusion that alpha-amylase is non-toxic upon repeated dermal exposure is supported by the knowledge of 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, 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.

- 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 1100 mg Total Organic Solids (TOS)/kg bw/day.

Based on repeated dose oral and dermal studies and weight of evidence, alpha-amylase 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 and dermal studies and weight of evidence, Alpha-Amylase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.