tert-butyl peracetate

Repeated dose toxicity: oral

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

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”)

Version / remarks:

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).

The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution in 1% CMC (aqueous) was demonstrated. The application formulations were considered to be stable for at least 4 hours and 7 days when kept refrigerated (about 4 °C).
In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.

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)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control, group 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

group 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

group 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

group 4

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):

In the highest dose group, all male and female animals were noted moving their head through the bedding material starting on day 3 of the pre-pairing period and continuing until the last administration. Some male and female animals were noted with ruffled fur starting on day 3 of the study continuing until necropsy. Between days 3 and 6 of the pre-pairing period, irregular breathing, ventral recumbency, hunched posture, uncoordinated gait and cold to touch were noted in all animals. Salivation prior to administration of test item was noted in some male and female animals starting on day 10 of the pre-pairing period until necropsy.
In the mid dose group, all animals started to move their heads through the bedding material starting on day 4 of the pre-pairing period until the end of study. On some days, a few animals were noted with salivation prior to administration of test item.
No treatment-related clinical signs were noted for the male and female animals in the lowest dose group. Only one female was noted with a black (bloody) right eye during gestation period that became milky during lactation period. This was considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

All female animals survived until scheduled necropsy.
In the highest dose group, males nos. 39 and 40 were found dead on day 14 (pre-pairing period), and male no. 32 was found dead on day 25 of the study (or day 3 of the after-pairing period). The cause of death in all three males was due to a pronounced ulceration of the fore stomach.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: In the high dose group mean body weight started to be statistically significantly decreased on day 2 of the pre-pairing period and continued to be statistically significantly decreased until the end of study
in the after pairing period. Mean body weight gain was statistically significantly decreased between day 2 and day 14 of the pre-pairing period, between days 4 to 8 of the pairing period, and between day 4 of the after pairing period until necropsy.
In the mid dose group, mean body weight was decreased starting on day 4 of the pre-pairing period and was statistically significantly decreased on days 13 and 14. During pairing period, the body weight was decreased (statistically significant on days 7 and 8). It stayed statistically significantly decreased for the whole after-pairing period. Mean body weight gain was statistically significantly decreased between day 9 and day 16 of the after-pairing period.
In the low dose group, mean body weight was slightly decreased starting during the pre-pairing period. It was statistically significantly decreased starting on day 4 of the after-pairing period until the end of study. Mean body weight gain was statistically significantly decreased on day 7, day 9 and 10 during the after-pairing period.
Females: In the high dose group, mean body weight started to be statistically significantly decreased on day 3 of the pre-pairing period and continued to be statistically significantly decreased until the end of study. Mean body weight gain was statistically significantly decreased between day 3 and day 14 of the pre-pairing period, and between days 2 to 21 of the gestation period.
In the mid dose group, mean body weight started to be statistically significantly decreased on day 3 of the pre-pairing period and continued to be statistically significantly decreased until day 13 of the gestation period. Mean body weight gain was only sporadically statistically significantly decreased on day 4 of the pre-pairing period.
In the low dose group, mean body weight was statistically significantly decreased on days 2, 3, 10, and 11 of the pre-pairing period. Mean body weight gain was considered to be not influenced by the treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: In the highest dose group, the mean food consumption was statistically significantly decreased during the prepairing period (-35.1%) and after-pairing period (-26.5%). Mean food consumption in mid and low dose group was considered to be not influenced during prepairing period. During the after-pairing period food consumption was statistically significantly decreased in mid dose group (-11.7%). In the low dose group, mean food consumption was statistically significantly decreased for days 1 - 8 of the after-pairing period. For the remaining period (days 8 - 20 of the after-pairing period), mean food consumption was not statistically significantly decreased.
Females: Mean food consumption in the highest dose group was statistically significantly decreased during pre-pairing (days 1 - 8, -43.2%), and gestation period (days 0 - 14, -23.0%). During lactation period, mean food consumption was only slightly decreased (-8.1%). Mean food consumption in mid and low dose groups was not influenced during pre-pairing, gestation and lactation periods.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

During pre-pairing period (day 13 to day 14), water consumption was measured as the animals of the high dose group seemed to consume more water than the animals in the other groups.
After a 24-hour time period, the male animals in the mid dose group were noted with an increased mean water intake (35.5 g/day). The mean water intake of the animals in the highest dose group was also measured at 35.3 g/day, when the male no. 38 was excluded (individual value of 4.8 g/day). The group mean value of control group was 32.2 g/day. In female animals, the mean water consumption was slightly increased in the highest dose group (30.4 g/day), when compared with the control (28.9 g/day). Since these differences were minimal a second waterconsumption measurement was not carried out.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In group 4 males, a tendency of inflammaty response was noted as increase of total leukocyte count, increase of absolute and relative neutrophils, and increase of platelets. In groups 2 and 3, none of the parameters under investigation for hematology was considered to be affected by exposure to the test item.
In males, the following parameters were statistically significantly increased in group 4: the mean hemoglobin concentration distribution width, the mean absolute reticulocyte count, the high fluorescence reticulocyte maturity index, the total leukocyte count, and the relative neutrophils. Mean relative reticulocyte count and the mean relative neutrophils were statistically significantly increased in groups 3 and 4. In groups 2, 3, and 4, the mean corpuscular volume and the mean corpuscular hemoglobin were statistically significantly increased. Statistically significantly decreased, only in group 4, were the mean erythrocyte count, hemoglobin, hematocrit, the mean corpuscular hemoglobin concentration, the reticulocyte maturity index (low fluorescence), the mean relative eosinophils and lymphocytes, and the mean activated partial thromboplastin time. Most of these findings were considered incidental as these statisticallysignificant values were within the range of the reference values. As the erythrocytes were lowered, a normal adjustment could be noticed as increases in reticulocytes and mean hemoglobin distribution width. In females, the following parameters were statistically significantly increased in group 4 the mean corpuscular volume, the mean red cell distribution width, the mean corpuscular hemoglobin, the mean hemoglobin concentration distribution width, and the mean absolute and relative reticulocyte count. Statistically significantly decreased in group 4 were the mean erythrocyte count, hemoglobin, hemoglobin concentration distribution width. As the erythrocytes were lowered, a normal adjustment could be noticed as raise in reticulocytes and mean hemoglobin distribution width. Most of these findings were considered incidental as these statistically significant values were covered by the reference values. In females, the mean absolute and relative reticulocyte count, the total leukocyte count as well as the mean absolute neutrophils and plateteles were noted with increased values in all groups including controls. As these parameters exceeded the reference values in all groups, these findings were considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the parameters under investigation for clinical biochemistry was considered to be affected by exposure to the test item. A few parameters in group 4 males, like cholesterol, aspartate aminotransferase, alkaline phosphatase, potassium, phosphorus, total protein, and globulins (also in group 3), were noted with statistically significantly values. Yet, these findings were considered incidental as the statistically significant values still were within the range of the reference values. Some parameters, like glucose, urea, and alanine aminotransferase, were increased in all groups and therefore considered incidental. In group 4 females, statistically significant mean values were noted for the parameters urea, bile acids, total cholesterol, alanine aminotransferase, total protein, and globulins. The parameter potassium was statistically significantly increased in groups 2, 3, and 4, and exceeded the reference values only in group 4. The parameters alanine aminotransferase and phosphorus exceeded the reference values in all groups. Yet, all these findings were considered incidental.

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Two of the parameters under investigation were considered to be affected by exposure to the test item during the functional observational battery. One male (no. 35) and two female (nos. 72 and 74) animals in the high dose group were noted with slight piloerection. One male (no. 35) animal was noted with reduced activity in the high dose group, and one female animal each (nos. 52, 72) was noted with reduced activity in groups low and high dose group. Slight piloerection in addition with reduced activity was considered test item related.
Two males in control, one male each in low and mid dose and two males in the high dose were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2, 1, and 2 males in groups contro, low and high dose group, respectively. A wet bedding in the home cage and many puddles of urine in the open field was noted for one male (no. 34) in the high dose. One female each in mid and high dose group was noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2, 1, 1, and 2 females in groups contro. low, mid and high dose, respectively. A wet bedding in the home cage and many puddles of urine in the open field was noted for one female (no. 65) in mid dose.
The hearing ability (Preyer’s reflex) was considered to be not influenced by treatment with the test item.

Body temperature (rectal) was dose-dependently reduced (statistically significantly in males of groups mid and high, and in females of high dose group. The decrease of body temperature was considered test item related as this finding corresponded to the clinical sign of “cold to touch” in the high dose.
Mean values for grip strength (fore- and hind paws) and landing foot splay were considered to be not influenced by treatment with the test item.

In the high dose group the reduction in locomotor activity, as assessed in terms of low beam counts in an activity monitor, was considered to be test item-related. The males were generally more active than the females. Still, in the high dose, the males were noted with a 31.5% reduction in activity and the females with a 26.4% reduction, when compared with the corresponding control group values. In low and mid dose group, locomotor activity was considered to be not influenced by treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of the experiment the substance induced adverse treatment-related changes in the stomach and small intestine, liver, bone marrow, spleen, lymphatic organs and in the male reproductive organs of group 4 animals (500 mg/kg/day). The stomach was also affected in animals of groups 2 and 3 (50 and 150 mg/kg/day), the duodenum and jejunum in animals of group 3, and in females the duodenum was also affected in group 2. Males nos. 32, 39 and 40 of group 4 died during the treatment period. The cause of death was due to a pronounced ulceration of the fore stomach.

Microscopically treatment-related changes were seen in the liver, spleen, stomach, small intestine, adrenal cortices, thymus (males only), mesenteric lymph nodes, Peyer’s patches and bone marrow. In the stomach the changes were noted in the fore stomach and consisted of acanthosis, parakeratosis, ulcerations, hemorrhage (one male of group 4) and inflammation.
One male rat of group 4 also presented a glandular mucosa hyperplasia. In the duodenum, jejunum and ileum (males only) mucosal hyperplasia was seen. Atrophy was seen in the thymus of males and in the Peyer’s patches of males and females. Lymphoid depletion was present in the spleen white pulp and in the mesenteric lymph nodes. Increased extramedullary hematopoiesis in the spleen and increased cellularity in the bone marrow were present in both sexes. Hemosiderin deposits and congestion were seen in the spleen of males and females. A high incidence of congestion was also seen in the mesenteric lymph nodes of males of group 4. Centrilobular hypertrophy of the hepatocytes was observed in males and females, and liver necrosis in one male animal. Hematopoiesis was seen in the liver of one male and female. A diffuse hypertrophy of the adrenal cortex and vacuolation were present in male and female rats. Apoptosis was reported in the adrenal cortex of one male rat. However it is likely that the changes in the adrenal cortex were secondary to the stress suffered by the animals of group 4.

In males reproductive organs treatment-related changes were seen in group 4 and consisted of tubular atrophy of the seminiferous tubules associated to germ cell depletion and spermatic giant cells, and in one animal to Sertoli cell vacuolation. In the epididymides of the same animals cellular debris was present whereas atrophy was seen in one animal. Atrophy of the prostate, seminal vesicles and coagulating glands was also present in a few animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

not affected

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Mating performance was considered to be not influenced by treatment with the test item. All mated females were pregnant. The fertility indices were 100.0% in all groups. The median pre-coital time was 3, 3, 2, and 4 days, and the mean precoital time was 3.2, 2.6, 2.3, and 3.7 days, respectively. The gestation length was considered to be not influenced by treatment with the test item. The mean duration of gestation was 21.4, 21.6, 21.3, and 21.9 days, respectively. The mean number of corpora lutea was considered to be likely not influenced by treatment with the test item. The mean number of corpora lutea was 20.4, 20.1, 20.3, and 17.6, respectively.

The implantation rate and the post implantation loss were considered to be likely not influenced by treatment with the test item. The mean implantation rate was 14.5, 13.9, 14.2, and 9.7, respectively. Thus, the implantation rate was considered reduced in the highest dose group. The total post implantation loss in control, low, mid and high dose group was 14, 6, 7, and 10, in 8, 4, 6, and 7 litters, respectively, corresponding to 9.7%, 4.3%, 4.9%, and 10.3% of implantations.

The number of living pups at first litter check was low in the high dose group. In control, low, mid and high dose group the total number of living pups was 131, 133, 135, and 87, in 10 litters each. In groups 1, 2, 3, and 4, the mean number of living pups was 13.1, 13.3, 13.5, and 8.7, respectively.

The post natal loss was statistically significantly increased in the high dose group. Thus, the viability index was statistically significantly decreased (90.8 in group 4 vs. 97.7 in group 1). In groups 1, 3, and 4, the postnatal loss was 3, 1, and 8 dead pups noted in 2, 1, and 3 litters, respectively. These data comply to 2.3%, 0.0%, 0.7% and 9.2% of dead pups during the postnatal period in groups 1, 2, 3, and 4, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

150 mg/kg bw/day

Based on:

test mat.

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.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

At first litter check and thereafter, no test item-related findings were noted. In litter 69 (group 3), one female pup was noted with a thread-like short tail and an anal atresia. It was killed for ethical reasons on day 1. This finding was considered incidental.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

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).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

At scheduled necropsy, no test item-related findings were noted. In litter 69 (group 3), one female pup (6F) was noted with a thread-like short tail and an anal atresia. This finding was considered incidental.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratios were considered to be not influenced by treatment with the test item. Sex ratios (% male/% female) were 47 / 53, 50 / 50, 43 / 57 and 43 / 57 in groups 1, 2, 3, and 4, respectively, at first litter check.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Based on these data, it can be concluded that the NOAEL for reproductive and developmental effects was 150 mg/kg bw/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).

Executive summary:

The substance 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). Dose levels were in terms of peroxide in the test item as supplied by the Sponsor. Therefore a correction factor of 2 was used for dose formulations. 

Treatment at 500 mg/kg bw/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 bw/day and 150 mg/kg bw/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 bw/day.

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. These were considered as secondary due to the severe maternal toxicity observed at the highest dose. Mean food consumption was -23.9% lower comparted to control in the pre-paring period in females at this dose. During the sensitive period of gestation (days 0-21 post coitum) mean food consumption was decreased by 17.8%. Body weight and body weight gain was also effected during gestation. Severe clinical symptoms were noted during gestation and lactation periods for all females of the high dose group.

Based on these data, it can be concluded that the NOAEL for reproductive and developmental effects was 150 mg/kg bw/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).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material information

Constituent 1

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 ethaneperoxoate

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).

The test item content in all samples was found to be within the accepted range of ±20% of the nominal content. In addition, the homogenous distribution in 1% CMC (aqueous) was demonstrated. The application formulations were considered to be stable for at least 4 hours and 7 days when kept refrigerated (about 4 °C).
In conclusion, the results obtained within this phase confirm the correct preparation and storage of application formulations during the conduct of this study.

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

Doses / concentrations 1

Remarks:

vehicle

Doses / concentrations 2

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (nominal)

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):

In the highest dose group, all male and female animals were noted moving their head through the bedding material starting on day 3 of the pre-pairing period and continuing until the last administration. Some male and female animals were noted with ruffled fur starting on day 3 of the study continuing until necropsy. Between days 3 and 6 of the pre-pairing period, irregular breathing, ventral recumbency, hunched posture, uncoordinated gait and cold to touch were noted in all animals. Salivation prior to administration of test item was noted in some male and female animals starting on day 10 of the pre-pairing period until necropsy.
In the mid dose group, all animals started to move their heads through the bedding material starting on day 4 of the pre-pairing period until the end of study. On some days, a few animals were noted with salivation prior to administration of test item.
No treatment-related clinical signs were noted for the male and female animals in the lowest dose group. Only one female was noted with a black (bloody) right eye during gestation period that became milky during lactation period. This was considered incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

All female animals survived until scheduled necropsy. In the highest dose group, males nos. 39 and 40 were found dead on day 14 (pre-pairing period), and male no. 32 was found dead on day 25 of the study (or day 3 of the after-pairing period). The cause of death in all three males was due to a pronounced ulceration of the fore stomach.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: In the high dose group mean body weight started to be statistically significantly decreased on day 2 of the pre-pairing period and continued to be statistically significantly decreased until the end of study in the after pairing period. Mean body weight gain was statistically significantly decreased between day 2 and day 14 of the pre-pairing period, between days 4 to 8 of the pairing period, and between day 4 of the after pairing period until necropsy.
In the mid dose group, mean body weight was decreased starting on day 4 of the pre-pairing period and was statistically significantly decreased on days 13 and 14. During pairing period, the body weight was decreased (statistically significant on days 7 and 8). It stayed statistically significantly decreased for the whole after-pairing period. Mean body weight gain was statistically significantly decreased between day 9 and day 16 of the after-pairing period.
In the low dose group, mean body weight was slightly decreased starting during the pre-pairing period. It was statistically significantly decreased starting on day 4 of the after-pairing period until the end of study. Mean body weight gain was statistically significantly decreased on day 7, day 9 and 10 during the after-pairing period.
Females: In the high dose group, mean body weight started to be statistically significantly decreased on day 3 of the pre-pairing period and continued to be statistically significantly decreased until the end of study. Mean body weight gain was statistically significantly decreased between day 3 and day 14 of the pre-pairing period, and between days 2 to 21 of the gestation period.
In the mid dose group, mean body weight started to be statistically significantly decreased on day 3 of the pre-pairing period and continued to be statistically significantly decreased until day 13 of the gestation period. Mean body weight gain was only sporadically statistically significantly decreased on day 4 of the pre-pairing period.
In the low dose group, mean body weight was statistically significantly decreased on days 2, 3, 10, and 11 of the pre-pairing period. Mean body weight gain was considered to be not influenced by the treatment with the test item.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: In the highest dose group, the mean food consumption was statistically significantly decreased during the prepairing period (-35.1%) and after-pairing period (-26.5%). Mean food consumption in mid and low dose group was considered to be not influenced during prepairing period. During the after-pairing period food consumption was statistically significantly decreased in mid dose group (-11.7%). In the low dose group, mean food consumption was statistically significantly decreased for days 1 - 8 of the after-pairing period. For the remaining period (days 8 - 20 of the after-pairing period), mean food consumption was not statistically significantly decreased.
Females: Mean food consumption in the highest dose group was statistically significantly decreased during pre-pairing (days 1 - 8, -43.2%), and gestation period (days 0 - 14, -23.0%). During lactation period, mean food consumption was only slightly decreased (-8.1%). Mean food consumption in mid and low dose groups was not influenced during pre-pairing, gestation and lactation periods.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

During pre-pairing period (day 13 to day 14), water consumption was measured as the animals of the high dose group seemed to consume more water than the animals in the other groups. After a 24-hour time period, the male animals in the mid dose group were noted with an increased mean water intake (35.5 g/day). The mean water intake of the animals in the highest dose group was also measured at 35.3 g/day, when the male no. 38 was excluded (individual value of 4.8 g/day). The group mean value of control group was 32.2 g/day. In female animals, the mean water consumption was slightly increased in the highest dose group (30.4 g/day), when compared with the control (28.9 g/day). Since these differences were minimal a second waterconsumption measurement was not carried out.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In group 4 males, a tendency of inflammaty response was noted as increase of total leukocyte count, increase of absolute and relative neutrophils, and increase of platelets. In groups 2 and 3, none of the parameters under investigation for hematology was considered to be affected by exposure to the test item.
In males, the following parameters were statistically significantly increased in group 4: the mean hemoglobin concentration distribution width, the mean absolute reticulocyte count, the high fluorescence reticulocyte maturity index, the total leukocyte count, and the relative neutrophils. Mean relative reticulocyte count and the mean relative neutrophils were statistically significantly increased in groups 3 and 4. In groups 2, 3, and 4, the mean corpuscular volume and the mean corpuscular hemoglobin were statistically significantly increased. Statistically significantly decreased, only in group 4, were the mean erythrocyte count, hemoglobin, hematocrit, the mean corpuscular hemoglobin concentration, the reticulocyte maturity index (low fluorescence), the mean relative eosinophils and lymphocytes, and the mean activated partial thromboplastin time. Most of these findings were considered incidental as these statisticallysignificant values were within the range of the reference values. As the erythrocytes were lowered, a normal adjustment could be noticed as increases in reticulocytes and mean hemoglobin distribution width. In females, the following parameters were statistically significantly increased in group 4 the mean corpuscular volume, the mean red cell distribution width, the mean corpuscular hemoglobin, the mean hemoglobin concentration distribution width, and the mean absolute and relative reticulocyte count. Statistically significantly decreased in group 4 were the mean erythrocyte count, hemoglobin, hemoglobin concentration distribution width. As the erythrocytes were lowered, a normal adjustment could be noticed as raise in reticulocytes and mean hemoglobin distribution width. Most of these findings were considered incidental as these statistically significant values were covered by the reference values. In females, the mean absolute and relative reticulocyte count, the total leukocyte count as well as the mean absolute neutrophils and plateteles were noted with increased values in all groups including controls. As these parameters exceeded the reference values in all groups, these findings were considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

None of the parameters under investigation for clinical biochemistry was considered to be affected by exposure to the test item. A few parameters in group 4 males, like cholesterol, aspartate aminotransferase, alkaline phosphatase, potassium, phosphorus, total protein, and globulins (also in group 3), were noted with statistically significantly values. Yet, these findings were considered incidental as the statistically significant values still were within the range of the reference values. Some parameters, like glucose, urea, and alanine aminotransferase, were increased in all groups and therefore considered incidental. In group 4 females, statistically significant mean values were noted for the parameters urea, bile acids, total cholesterol, alanine aminotransferase, total protein, and globulins. The parameter potassium was statistically significantly increased in groups 2, 3, and 4, and exceeded the reference values only in group 4. The parameters alanine aminotransferase and phosphorus exceeded the reference values in all groups. Yet, all these findings were considered incidental.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Two of the parameters under investigation were considered to be affected by exposure to the test item during the functional observational battery. One male (no. 35) and two female (nos. 72 and 74) animals in the high dose group were noted with slight piloerection. One male (no. 35) animal was noted with reduced activity in the high dose group, and one female animal each (nos. 52, 72) was noted with reduced activity in groups low and high dose group. Slight piloerection in addition with reduced activity was considered test item related.
Two males in control, one male each in low and mid dose and two males in the high dose were noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2, 1, and 2 males in groups contro, low and high dose group, respectively. A wet bedding in the home cage and many puddles of urine in the open field was noted for one male (no. 34) in the high dose. One female each in mid and high dose group was noted with a decreased rearing (n<3). A few or small puddles of urine were noted for 2, 1, 1, and 2 females in groups contro. low, mid and high dose, respectively. A wet bedding in the home cage and many puddles of urine in the open field was noted for one female (no. 65) in mid dose.
The hearing ability (Preyer’s reflex) was considered to be not influenced by treatment with the test item.

Body temperature (rectal) was dose-dependently reduced (statistically significantly in males of groups mid and high, and in females of high dose group. The decrease of body temperature was considered test item related as this finding corresponded to the clinical sign of “cold to touch” in the high dose.
Mean values for grip strength (fore- and hind paws) and landing foot splay were considered to be not influenced by treatment with the test item.

In the high dose group the reduction in locomotor activity, as assessed in terms of low beam counts in an activity monitor, was considered to be test item-related. The males were generally more active than the females. Still, in the high dose, the males were noted with a 31.5% reduction in activity and the females with a 26.4% reduction, when compared with the corresponding control group values. In low and mid dose group, locomotor activity was considered to be not influenced by treatment with the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The absolute and relative weights of the liver were increased in males and females of group 4, and in males of group 3 the relative liver weight was increased mainly due to animal no. 22. The absolute and relative weights of the adrenal glands and spleen were increased in females of group 4 while the relative weight of these organs was increased in males of group 4. The absolute and relative weights of the thymus were decreased in males of group 4. The epididymides’ weight was statistically significantly decreased while the relative epididymides’ weight was statistically significantly increased. There were no statistically significant changes in all other mean organ weights. The relative weights of brain, heart, kidneys, adrenals, and spleen were statistically significantly increased in group 4 males. Mean testes weight ratio was statistically significantly increased in groups 3 and 4.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In group 4, three males (nos. 32, 39, and 40) were found dead prior to the scheduled necropsy. Male no. 32 was noted with an advanced autolysis, males nos. 39 and 40 were noted with a dark red discolouration of the lungs. Males that were examined during the scheduled necropsy were found with a thickened mucosa of the fore stomach (nos. 33, 34, 36, 37), gray white, firm nodules in the stomach wall (no. 35), a dilated duodenum (nos. 31, 33, 34, 35, 36, 37, 38), reddish discoloured mesenteric lymph nodes (nos. 33, 34, 35), or a thymus reduced in size (nos. 31, 33, 35, 36, 38). In females, the spleen was noted with a fibrin-like coating (nos. 71, 73, 74) or was enlarged (no. 75), the fore stomach showed a red brown mucosa (no. 74), the mucosa of duodenum and jejunum was thickened (nos. 73, 74), the mesenteric lymph node was dark red discoloured (no. 73), or the diaphragm was adherent to the stomach (no. 74). All male and female animals in groups 2 and 3 were noted without any treatment-related macroscopically findings during necropsy. Female no. 57 (in group 2) was noted with a gray white discoloured right eye that was considered incidental.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of the experiment the substance induced adverse treatment-related changes in the stomach and small intestine, liver, bone marrow, spleen, lymphatic organs and in the male reproductive organs of group 4 animals (500 mg/kg/day). The stomach was also affected in animals of groups 2 and 3 (50 and 150 mg/kg/day), the duodenum and jejunum in animals of group 3, and in females the duodenum was also affected in group 2. Males nos. 32, 39 and 40 of group 4 died during the treatment period. The cause of death was due to a pronounced ulceration of the fore stomach.

Microscopically treatment-related changes were seen in the liver, spleen, stomach, small intestine, adrenal cortices, thymus (males only), mesenteric lymph nodes, Peyer’s patches and bone marrow. In the stomach the changes were noted in the fore stomach and consisted of acanthosis, parakeratosis, ulcerations, hemorrhage (one male of group 4) and inflammation.
One male rat of group 4 also presented a glandular mucosa hyperplasia. In the duodenum, jejunum and ileum (males only) mucosal hyperplasia was seen. Atrophy was seen in the thymus of males and in the Peyer’s patches of males and females. Lymphoid depletion was present in the spleen white pulp and in the mesenteric lymph nodes. Increased extramedullary hematopoiesis in the spleen and increased cellularity in the bone marrow were present in both sexes. Hemosiderin deposits and congestion were seen in the spleen of males and females. A high incidence of congestion was also seen in the mesenteric lymph nodes of males of group 4. Centrilobular hypertrophy of the hepatocytes was observed in males and females, and liver necrosis in one male animal. Hematopoiesis was seen in the liver of one male and female. A diffuse hypertrophy of the adrenal cortex and vacuolation were present in male and female rats. Apoptosis was reported in the adrenal cortex of one male rat. However it is likely that the changes in the adrenal cortex were secondary to the stress suffered by the animals of group 4.

In males reproductive organs treatment-related changes were seen in group 4 and consisted of tubular atrophy of the seminiferous tubules associated to germ cell depletion and spermatic giant cells, and in one animal to Sertoli cell vacuolation. In the epididymides of the same animals cellular debris was present whereas atrophy was seen in one animal. Atrophy of the prostate, seminal vesicles and coagulating glands was also present in a few animals.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

no

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. Please refer to IUCLID Section 7.8.1 for details.

Applicant's summary and conclusion

Conclusions:

Data obtained from the Combined Repeated-Dose and Reproduction / Developmental Screening Study in rats revealed an oral NOAEL of 50 mg/kg bw/day calculated as peroxide for parental effects. Therefore, the NOAEL is expected to be 100 mg/kg bw/day for the test material (50% act. ingr. in Shellsol T).

Executive summary:

The substance 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). 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.

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. These were considered as secondary due to the severe maternal toxicity observed at the highest dose. Mean food consumption was -23.9% lower comparted to control in the pre-paring period in females at this dose. During the sensitive period of gestation (days 0-21 post coitum) mean food consumption was decreased by 17.8%. Body weight and body weight gain was also effected during gestation. Severe clinical symptoms were noted during gestation and lactation periods for all females of the high dose group.

Based on these data, it can be concluded that the NOAEL for parental effects based on histological findings in the gastro-intestinal tract. Therefore, the NOAEL is expected to be 100 mg/kg bw/day for the test material (50% act. ingr. in Shellsol T)