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Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-03-14 and 2008-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
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
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: (P) x wks: males 8 weeks, females 10 weeks
- Weight at study initiation: (P) Males: 240 - 280 g; Females: 175 - 215 g
- Housing: in Makrolon cages (type-3) with wire mesh tops and standard granulated softwood bedding (Lignocel, Schill AG, 4132 Muttenz/Switzerland); pre-pairing period: individually housing; pairing period: one male/one female in Makrolon pairing cages; at the end of the pairing period: individually housing; during the lactation period (until day 4 of lactation): dams were housed together with their litters
- 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
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on 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)

All animals received a dose volume of 10 mL/kg body weight with a daily adjustment of the individual volume to the actual body weight. Control animals were dosed with the vehicle alone.

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

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until mating (maximum 14 days)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
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: throughout the pre-pairing, the pairing, the gestation and the lactation periods until day 4 post partum (last dosing) = 54 days in total
Frequency of treatment:
once per day
Details on study schedule:
see information on materials and methods (table 1)
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:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: 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

OTHER: Functional observational battery, mortality rate, organ weight

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.
Oestrous cyclicity (parental animals):
The copulation plug was observed, and/or the daily vaginal smear was sperm-positive.
Sperm parameters (parental animals):
testes weight, epididymis weight
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
litter size, live births, postnatal mortality (0 - 4 post partum), abnormal findings (until day 4 post partum), sex ratios, pup weights (to day 4 post partum), necropsy findings, body weight (on day 0, 1 and 4 post partum)

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
Males were sacrificed after the first dams had reached day 4 post partum. Females were sacrificed on day 5 post partum after blood sampling. Males and females were killed by exsanguination following an intraperitoneal injection of sodium pentobarbital. The animals were examined macroscopically for any structural abnormalities or pathological changes, with special attention paid to the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible haemorrhagic areas of implantation sites.
Postmortem examinations (offspring):
- Pups were sacrificed on day 4 post partum. Pups were killed by an intraperitoneal injection of sodium pentobarbital.
- Dead pups (except if excessively cannibalized) and pups killed at day 4 of lactation were examined macroscopically.
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.
Reproductive indices:
females mating index, fertility index, conception index, gestation index
The formulas for calculation can be found in table 2 in "Any other information on materials and methods"
Offspring viability indices:
birth index and viability index
The formulas for calculation can be found in table 2 in "Any other information on materials and methods"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
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
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): 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 FOOD CONSUMPTION (PARENTAL ANIMALS): Body weight was dose-dependently decreased in both gender. Mean food consumption was decreased in female animals at 500 mg/kg during pre-pairing and gestation period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): not examined

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS): not affected

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): not examined

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): not affected

ORGAN WEIGHTS (PARENTAL ANIMALS): 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 (PARENTAL ANIMALS): 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 (PARENTAL ANIMALS): 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 (PARENTAL ANIMALS): 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.
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Based on:
other: peroxide
Sex:
male/female
Basis for effect level:
other: 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.
Remarks on result:
other: Generation: P/F1 (migrated information)
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: Generation: P/F1 (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
see details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING): The post natal loss was statistically significantly increased at 500 mg/kg bw/day. Thus, the viability index was statistically significantly decreased (90.8 in group 4 (500 mg/kg bw/day) vs. 97.7 in control group).

CLINICAL SIGNS (OFFSPRING): no effects

BODY WEIGHT (OFFSPRING): Slightly lower pup body weights at first litter check were noted for male and female pups at 500 mg/kg bw/day. Mean body weight gain in surviving group 4 (at 500 mg/kg bw/day) pups until day 4 post partum was lowered (12,5%) when compared with the group 1 (control) pups. These findings were considered likely test item related.

SEXUAL MATURATION (OFFSPRING): not examined

ORGAN WEIGHTS (OFFSPRING): not examined

GROSS PATHOLOGY (OFFSPRING): no effects

HISTOPATHOLOGY (OFFSPRING): not examined

Remarks on result:
other: see "Details on results"
Reproductive effects observed:
not specified
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 based on the active ingredient. Therefore, the NOAEL for the test material (50% active ingredient in aliphatic hydrocarbon solvent) is calculated to be 300 mg/kg bw/day.
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was to generate information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. This study was in accordance to the OECD guideline 422.

TBPA was administered once daily orally (by gavage) at dosages of 0, 50, 150, and 500 mg/kg/day, 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). A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (1% carboxymethylcellulose).

Concerning the reproduction data, the fertility index was 100% in all groups. 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. 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 based on the active ingredient. Therefore, the NOAEL for the test material (50% act. ingr. in aliphatic hydrocarbon solvent) is calculated to be 300 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study is considered acceptable without restrictions as it was conducted according to GLP regulations and OECD guideline.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test was to generate information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition. This study was in accordance to the OECD guideline 422.

TBPA was administered once daily orally (by gavage) at dosages of 0, 50, 150, and 500 mg/kg/day, 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). A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (1% carboxymethylcellulose).

Concerning the reproduction data, the fertility index was 100% in all groups. 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. 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 calculated as peroxide. Therefore, the NOAEL is calculated to be 300 mg/kg bw/day for the test material (50% tert-butylperacetate in aliphatic hydrocarbon solvent).


Short description of key information:
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 based on the active ingredient. Therefore, the NOAEL is calculated to be 300 mg/kg bw/day for the test material (50% tert-butylperacetate in aliphatic hydrocarbon solvent).

Justification for selection of Effect on fertility via oral route:
Only one study available.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results, the test substance is not considered to be classified for reproductive toxicity according to Regulation (EC) No 1272/2008.

Additional information