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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-14 and 2008-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
1996
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl peracetate
EC Number:
203-514-5
EC Name:
tert-butyl peracetate
Cas Number:
107-71-1
Molecular formula:
C6H12O3
IUPAC Name:
tert-butyl peracetate
Details on test material:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in the refrigeratore in the dark between +10 and +30°C

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation: males (8 weeks), females (10 weeks)
- Weight at study initiation: Males: 240 - 280 grams, Females: 175 - 215 grams
- Fasting period before study: none
- Housing: Animals were housed in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland).
- Diet: Pelleted standard Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst/Switzerland) was available ad libitum (Batch No. 67/06).
- Water: Tap water from Füllinsdorf in bottles was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/ 3
- Humidity (%): 30 -70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
1%, medium viscosity
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle were added (w/v). Using an appropriate homogenizer a homogeneous mixture was prepared. Having obtained a homogeneous mixture, vehicle was added until the required final volume was achieved. Separate formulations were prepared for each concentration. During the daily administration period homogeneity of the test item in the vehicle was maintained using a magnetic stirrer.
Frequency of dose formulation: weekly, Storage of dose formulations: refrigerator (2 - 8 °C)

VEHICLE
- Justification for use and choice of vehicle: 1% CMC was used as the vehicle for the test item in the dose groups and was administered as the control item to the animals of the control group. The test item was miscible in saturated aliphatic and aromatic solvents, immiscible in water at 10°C.
- Lot/batch no.: 1119535 24604357
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples for determination of concentration, homogeneity and stability (4 hours and 7 days) of the dose formulations were taken during the first week of the administration period. Additionally, samples for determination of concentration and homogeneity were taken during the last week of the administration period. The test item concentrations were determined by HPLC coupled to an UV/VIS detector (VWR-Hitachi L-2400).
Duration of treatment / exposure:
Males: 42 days
Females: from 14 days prior to mating until day 4 post partum (54 days in total)
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 150, 500 mg/kg bw/day
Basis:
other: as peroxide
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
The rat is a suitable rodent species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.
Dose levels were selected in agreement with the Sponsor, based on the results of a preliminary dose range finding study (RCC Study No. A42390), where 600 mg/kg/ day as peroxide was used as highest dose level.
Positive control:
no data

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- daily
- Cage side observations checked: unusual body movements, abnormal behavior and posture as well as resistance to removal

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once prior to the first test item administration and weekly thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Mean daily food consumption were calculated by a computer program based on on-line record data.

COMPOUND INTAKE: not examined

FOOD EFFICIENCY: not examined

WATER CONSUMPTION AND COMPOUND INTAKE: not examined

OPHTHALMOSCOPIC EXAMINATION: not examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: on the day before or on the day of scheduled necropsy, females: on day 5 post partum
- Anaesthetic used for blood collection: Yes, light isoflurane anaesthesia
- Animals fasted: Yes (overnight)
- Parameters checked: Erythrocyte count, Haemoglobin, Haematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Haemoglobin concentration distribution width, Platelet count, Total leukocyte count, Differential leukocyte count, Coagulation: Thromboplastin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: on the day before or on the day of scheduled necropsy, females: on day 5 post partum
- Anaesthetic used for blood collection: Yes, light isoflurane anaesthesia
- Animals fasted: Yes (overnight)
- Parameters checked: Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Aspartate aminotransferase, Alanine aminotransferase, Bile acids, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein (total), Albumin, Globulin, Albumin/Globulin ratio

URINALYSIS: not examined

NEUROBEHAVIOURAL EXAMINATION: not examined

OTHER: organ weight, mortality rate, functional observational battery
Sacrifice and pathology:
Males were sacrificed after the first dams had reached day 4 post partum. Females were sacrificed on day 5 post partum after blood sampling.
GROSS PATHOLOGY: yes
HISTOPATHOLOGY: yes

Organ weights were determined from the following organs for five adult males and females (wet weight):
liver, spleen, adrenals*, brain, thymus, heart, kidneys* (*weight as pairs)
The testes* and epididymides* of all parental males were weighed.

Of all parental males the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: prostate, testes (in Bouin's fixative), seminal, vesicles with coagulation gland and epididymides (in Bouin's fixative). Of all parental females ovaries were taken.

In addition, of the five males and females per group selected for organ weights, the following tissues were preserved in neutral phosphate buffered 4% formaldehyde solution: gross lesions, heart, brain, thymus, spinal cord, thyroid, small and large intestines (incl. Peyer's patches), trachea and lungs (preserved by inflation with fixative), stomach, uterus (with vagina), liver, urinary bladder, kidneys, lymph nodes (mandibular, mesenterial), adrenals, peripheral nerve, spleen and bone marrow.
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett t-test, based on a pooled variance estimate, was used for inter-group comparisons.
- The Steel test (rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY: 500 mg/kg/day: 3 dead males, clinical signs in all animals (ruffled fur, irregular breathing, ventral recumbency, uncoordinated gait, cold to touch); 500 mg/kg/day and 150 mg/kg/day: signs of discomfort in all animals in the way that the animals moved their heads through the bedding material after the daily administration of test item

BODY WEIGHT AND WEIGHT GAIN: dose-dependently decreased in both genders

FOOD CONSUMPTION: Mean food consumption was decreased in female animals at 500 mg/kg during pre-pairing and gestation period. Compound intake was not examined.

HAEMATOLOGY: In males, a tendency of inflammatory response could be noted as increase of total leukocyte count, increase of absolute and relative neutrophils, and increase of platelets at 500 mg/kg.

CLINICAL CHEMISTRY: A few parameters were noted with statistically significantly values still within the range of the reference values. Some parameters were increased in all groups. Therefore, none of the parameters under investigation for clinical biochemistry was considered to be affected by exposure to the test item.

ORGAN WEIGHTS: In male animals of group 4 (500 mg/kg bw), the mean liver weight was statistically significantly increased. But for organ/body weight ratio, brain, heart, kidneys, adrenals, and spleen were statistically significantly increased in group 4 (500 mg/kg bw).The epididymides’ weight was statistically significantly decreased while the relative epididymides’ weight was statistically significantly increased. Mean testes weight ratio was statistically significantly increased in groups 3 (150 mg/kg bw) and 4 (500 mg/kg bw). In female animals of group 4 (500 mg/kg bw), the mean organ weights of liver, adrenals, and spleen were statistically significantly increased.

GROSS PATHOLOGY: at 500 mg/kg/day, the gastro-intestinal organs were noted with signs of adaptive defense (thickened mucosa, altered mesenteric lymph nodes), fibrin-like coatings or adherences

HISTOPATHOLOGY: adverse treatment-related changes noted in stomach and small intestine, liver, bone marrow, spleen, lymphatic organs, and in the males reproductive organs at 500 mg/kg/day; stomach was also affected in animals at 150 and 50 mg/kg/day, the duodenum and jejunum in animals at 150 mg/kg/day at 50 mg/kg/day

OTHER FINDINGS: Behavioral investigations (slight piloerection, reduced activity, reduced rectal body temperature, slightly reduced locomotor activity) were considered to be influenced by treatment at 500 mg/kg.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: histological findings in the gastrotestinal tract
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histological findings in the gastrointestinal tract

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Effects were noted on reproduction data at 500 mg/kg/day as reduction of implantations, small number of live pups/dam at birth and at day 4 of lactation, smaller litter weight and slightly smaller pup weight.

Applicant's summary and conclusion

Conclusions:
Based on these data, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for reproductive and developmental effects was 150 mg/kg body weight/day; and 50 mg/kg/day calculated as peroxide for parental effects based on histological findings in the gastro-intestinal tract. Therefore, the NOAEL of the test material (50% in Shellsol T) is expected to be 100 mg/kg/day for parental effects.
Executive summary:

TBPA was administered once daily orally (by gavage) at dosages of 0, 50, 150, and 500 mg/kg/day calculated as peroxide, to male rats for 42 days in total and to female rats throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post partum (last dosing). Treatment at 500 mg/kg/day was associated with death of three males, and severe clinical symptoms in all animals (ruffled fur, irregular breathing, ventral recumbency, uncoordinated gait, cold to touch). A sign of discomfort was noted in all animals at 500 mg/kg/day and 150 mg/kg/day in the way that the animals moved their heads through the bedding material after the daily administration of test item. Mean food consumption was decreased in female animals at 500 mg/kg during pre-pairing and gestation period. In males, mean food consumption was dose-dependently decreased at all test item treated groups. Mean body weights were dose-dependently decreased in both gender. Behavioral investigations (slight piloerection, reduced activity, reduced rectal body temperature, slightly reduced locomotor activity) were considered to be influenced by treatment at 500 mg/kg. In males, a tendency of inflammatory response could be noted as increase of total leukocyte count, increase of absolute and relative neutrophils, and increase of platelets at 500 mg/kg. After treatment at 500 mg/kg/day, the gastro-intestinal organs were noted with signs of adaptive defense (thickened mucosa, altered mesenteric lymph nodes), fibrin-like coatings or adherences. Treatment at 500 mg/kg/day was associated with increased liver and kidney weights. Histopathological effects were noted in stomach and small intestine, liver, bone marrow, spleen, lymphatic organs, and in the males reproductive organs at 500 mg/kg/day. The stomach was also affected in animals at 150 and 50 mg/kg/day, the duodenum and jejunum in animals at 150 mg/kg/day and the duodenum in females at 50 mg/kg/day. Based on these data, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for parental effects was 50 mg/kg/day calculated as peroxide based on histological findings in the gastro-intestinal tract. Therefore, the NOAEL is expected to be 100 mg/kg/day for the test material (50% act. ingr. in Shellsol T).